CN102220376B - Adeno associated virus (AAV) is evolved, sequence, carrier containing these sequences and their application - Google Patents

Abstract

The present invention provides novel capsid and vector sequences of adeno-associated virus and host cells containing these sequences. Methods for producing rAAV particles using such host cells and vectors are also described. AAV-mediated delivery of therapeutic and immunogenic genes using the vectors of the invention is also provided.

Description

Adeno-Associated Virus (AAV) Clades, Sequences, Vectors Containing These Sequences, and Their Applications

The patent application of the present invention is that the international application number is PCT/US2004/028817, the international application date is September 30, 2004, the application number entering the Chinese national stage is "200480027905.2", and the invention name is "adeno-associated virus (AAV) clade , sequences, vectors containing these sequences and their applications” is a divisional application of the invention patent application.

Statement Regarding Federally Sponsored Research or Development

This application includes work funded by grants NIDDK P30DK47757 and NHLBI P01HL59407 from the National Institutes of Health (NIH). The US Government has certain rights in this invention.

Background of the invention

Adeno-associated virus (AAV), a member of the Parvoviridae family, is a small non-enveloped, icosahedral virus with a single-stranded linear DNA genome of 4.7 kilobases (kb) to 6 kb. Since the virus was found as a contaminant of a purified adenovirus stock, AAV was assigned the genus Dependovirus. The AAV life cycle includes a latent period (site-specific integration of the AAV genome into the host chromosome following infection), an infectious period (after adenovirus or herpes simplex virus infection, the integrated genome is then rescued, replicated, and packaged into infectious virus) . The non-pathogenic properties, wide host range of infectivity (including non-dividing cells) and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.

Recent studies have shown that AAV vectors are the preferred vehicles for gene delivery. To date, several well-characterized different AAVs have been isolated from humans or non-human primates (NHPs).

It has been found that AAV of different serotypes exhibits different transfection efficiencies, and also exhibits tropism to different cells or tissues. However, the relationship between these different serotypes has not been studied before.

AAV-based constructs are ideal for the delivery of heterologous molecules.

Summary of the invention

The present invention provides a "superfamily" or "clade" of phylogenetically related sequences of AAV. These AAV clades provide a source of AAV sequences for targeting and/or delivering molecules to desired target cells or tissues.

One aspect of the invention provides an AAV clade having at least three AAV members based on an alignment of the AAV vp1 amino acid sequences using a Neighbor-Joining heuristic algorithm with a self-citation value of at least 75% (based on at least 1000 replicates). heuristic) with a Poisson correction distance measurement of no greater than 0.05 was determined to be phylogenetically correlated. A suitable AAV clade consists of the AAV sequences used to generate the vector.

The invention also provides previously unknown human AAV serotypes, referred to herein as clone 28.4/hu.14 or AAV serotype 9. Therefore, another aspect of the present invention provides an AAV of serotype 9 consisting of an AAV capsid serologically related to the capsid of the sequence of amino acids 1-736 of SEQ ID NO: 123, serologically is different from any capsid protein in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.

Vectors constructed with this capsid of huAAV9 exhibited gene transfer efficiencies similar to AAV8 in liver, better than AAV in muscle and 200-fold higher than AAV5 in lung. Furthermore, this new human AAV serotype has less than 85% sequence identity with previously described AAV1 to AAV8 and is not cross-neutralized by any of these AAVs.

The invention also provides other novel AAV sequences, compositions containing these sequences and their uses. Advantageously, these compositions are particularly useful for AAV vectors that require repeated administration for therapeutic or prophylactic purposes.

These and other aspects of the invention will become apparent from the following detailed description of the invention.

Brief description of the drawings

Figure 1 is a phylogenetic tree constructed using the adjacency heuristic with Poisson-corrected distance measures. Relationships were determined based on isolated AAV vp1 capsid proteins and isolated AAVs grouped in clades. Groups of individual capsid clones are classified in clades based on their common ancestry. Clades are named A through F; subtypes are indicated by the clade letter plus a numerical value.

Figure 2A-2AE compares the amino acid sequence of the AAV vp1 capsid protein of the present invention (the numbering of each sequence is published) with the previously published AAV1 [SEQ ID NO: 219], AAV2 [SEQ ID NO: 221], AAV3-3 [SEQ ID NO:217], AAV4-4[SEQ ID NO:218], AAV5[SEQ ID NO:216], AAV6[SEQ ID NO:220], AAV7[SEQ ID NO:222], AAV8[SEQ ID NO: 223] with rh.25/42-15, 29.3/bb.1, cy.2, 29.5/bb.2, rh.32, rh.33, rh.34, rh.10, rh.24, rh14 , rh.16, rh.17, rh.12, rh.18, rh.21 (formerly 41.10), rh.25 (formerly 41.15), rh2, rh.31, cy.3, cy.5 , rh.13, cy.4, cy.6, rh.22, rh.19, rh.35, rh.37, rh.36, rh.23, rh.8, and ch5 [Published U.S. Patent Application No. 2003 /0138772Al (July 24, 2003)]. Sequences of the present invention include hu.14/AAV9 [SEQ ID NO: 123], hu.17 [SEQ ID NO: 83], hu.6 [SEQ ID NO: 84], hu.42 [SEQ ID NO: 85] , hu.38[SEQ ID NO:86], hu.40[SEQ ID NO:87], hu.37[SEQ ID NO:88], rh.40[SEQ ID NO:92], rh.52[SEQ ID NO:96], rh.53[SEQ ID NO:97], rh.49[SEQ ID NO:103], rh.51[SEQ ID NO:104], rh.57[SEQ ID NO:105], rh. 58[SEQ ID NO:106], rh.61[SEQ ID NO:107], rh.50[SEQ ID NO:108], rh.43[SEQ ID NO:163], rh.62[SEQ ID NO:114 ], rh.48[SEQ ID NO:115], 4-9/rh.54[SEQ ID NO:116] and 4-19/rh.55[SEQ ID NO:117], hu.31[SEQ ID NO :121], hu.32[SEQ ID NO:122], hu.34[SEQ ID NO:125], hu.45[SEQ ID NO:127], hu.47[SEQ ID NO:128], hu.13 [SEQ ID NO:129], hu.28[SEQ ID NO:130], hu.29[SEQ ID NO:132], hu.19[SEQ ID NO:133], hu.20[SEQ ID NO:134], hu.21[SEQ ID NO:135], hu.23.2[SEQ ID NO:137], hu.22[SEQ ID NO:138], hu.27[SEQ ID NO:140], hu.4[SEQ ID NO :141], hu.2[SEQ ID NO:143], hu.1[SEQ ID NO:144], hu.3[SEQ ID NO:145], hu.25[SEQ ID NO:146], hu.15 [SEQ ID NO:147], hu.16[SEQ ID NO:148], hu.18[SEQ ID NO:149], hu.7[SEQ ID NO:150], hu.11[SEQ ID NO:153], hu.9[SEQ ID NO:155], hu.10[SEQ ID NO:156], hu.48[SEQ ID NO: 157], hu.44[SEQ ID NO:158], hu.46[SEQ ID NO:159], hu.43[SEQ ID NO:160], hu.35[SEQ ID NO:164], hu.24[ SEQ ID NO:136], rh.64[SEQ ID NO:99], hu.41[SEQ ID NO:91], hu.39[SEQ ID NO:102], hu.67[SEQ ID NO:198], hu.66[SEQ ID NO:197], hu.51[SEQ ID NO:190], hu.52[SEQ ID NO:191], hu.49[SEQ ID NO:189], hu.59[SEQ ID NO: 192], hu.57[SEQ ID NO:193], hu.58[SEQ ID NO:194], hu.63[SEQ ID NO:195], hu.64[SEQ ID NO:196], hu.60[ SEQ ID NO:184], hu.61[SEQ ID NO:185], hu.53[SEQ ID NO:186], hu.55[SEQ ID NO:187], hu.54[SEQ ID NO:88], hu.6 [SEQ ID NO: 84] and rh.56 [SEQ ID NO: 152]. These capsid sequences are also found in the Sequence Listing incorporated by reference.

Figure 3A-3CN compares the nucleic acid sequence of the AAV vp1 capsid protein of the present invention (the numbering of each sequence is published) with the previously published AAV5 (SEQ ID NO: 199), AAV3-3 (SEQ ID NO: 200), AAV4 -4 (SEQ ID NO: 201), AAV1 (SEQ ID NO: 202), AAV6 (SEQ ID NO: 203), AAV2 (SEQ ID NO: 211), AAV7 (SEQ ID NO: 213) and AAV8 (SEQ ID NO: 213) NO: 214), rh.25/42-15, 29.3/bb.1, cy.2, 29.5/bb.2, rh.32, rh.33, rh.34, rh.10, rh.24, rh14 , rh.16, rh.17, rh.12, rh.18, rh.21 (formerly known as 41.10), rh.25 (formerly known as 41.15; GenBank accession number AY530557), rh2, rh.31, cy. 3. cy.5, rh.13, cy.4, cy.6, rh.22, rh.19, rh.35, rh.36, rh.37, rh.23, rh.8 and ch.5[ Published US Patent Application No. 2003/0138772A1 (July 24, 2003)]. The nucleic acid sequences of the present invention include hu.14/AAV9 (SEQ ID NO: 3), LG-4/rh.38 (SEQ ID NO: 7), LG-10/rh.40 (SEQ ID NO: 14), N721-8/ rh.43 (SEQ ID NO: 43), 1-8/rh.49 (SEQ ID NO: 25), 2-4/rh.50 (SEQ ID NO: 23), 2-5/rh.51 (SEQ ID NO : 22), 3-9/rh.52 (SEQ ID NO: 18), 3-11/rh.53 (SEQ ID NO: 17), 5-3/rh.57 (SEQ ID NO: 26), 5- 22/rh.58 (SEQ ID NO: 27), 2-3/rh.61 (SEQ ID NO: 21), 4-8/rh.64 (SEQ ID NO: 15), 3.1/hu.6 (SEQ ID NO : 5), 33.12/hu.17 (SEQ ID NO: 4), 106.1/hu.37 (SEQ ID NO: 10), LG-9/hu.39 (SEQ ID NO: 24), 114.3/hu.40 ( SEQ ID NO: 11), 127.2/hu.41 (SEQ ID NO: 6), 127.5/hu.42 (SEQ ID NO: 8) and hu.66 (SEQ ID NO: 173), 2-15/rh.62 (SEQ ID NO:33), 1-7/rh.48 (SEQ ID NO:32), 4-9/rh.54 (SEQ ID NO:40), 4-19/rh.55 (SEQ ID NO:37) , 52/hu.19 (SEQ ID NO: 62), 52.1/hu.20 (SEQ ID NO: 63), 54.5/hu.23 (SEQ ID NO: 60), 54.2/hu.22 (SEQ ID NO: 67), 54.7/hu.24 (SEQ ID NO:66), 54.1/hu.21 (SEQ ID NO:65), 54.4R/hu.27 (SEQ ID NO:64), 46.2/hu.28 (SEQ ID NO:68 ), 46.6/hu.29 (SEQ ID NO: 69), 128.1/hu.43 (SEQ ID NO: 80), 128.3/hu.44 (SEQ ID NO: 81) and 130.4/hu.48 (SEQ ID NO: 78), 3.1/hu.9 (SEQ ID NO: 58), 16.8/hu.10 (SEQ ID NO: 56), 16.12/hu.11 (SEQ ID NO: 57), 145.1/hu.53 (SEQ ID NO : 176), 145.6/hu.55 (SEQ ID NO: 178), 145.5/hu.54 (SEQ ID NO: 177), 7.3/hu.7 (SEQ ID NO: 55), 52/hu.19 (SEQ ID NO: 62), 33.4/hu.15 (SEQ ID NO: 50), 33.8/hu.16 (SEQ ID NO: 51), 58.2/hu.25 (SEQ ID NO: 49), 161.10/hu.60 ( SEQ ID NO: 170), H-5/hu.3 (SEQ ID NO: 44), H-1/hu.1 (SEQ ID NO: 46), 161.6/hu.61 (SEQ ID NO: 174), hu. 31 (SEQ ID NO: 1), hu.32 (SEQ ID NO: 2), hu.46 (SEQ ID NO: 82), hu.34 (SEQ ID NO: 72), hu.47 (SEQ ID NO: 77 ), hu.63 (SEQ ID NO: 204), hu.56 (SEQ ID NO: 205), hu.45 (SEQ ID NO: 76), hu.57 (SEQ ID NO: 206), hu.35 (SEQ ID NO: 206), hu.35 (SEQ ID NO: 205), hu. NO: 73), hu.58 (SEQ ID NO: 207), hu.51 (SEQ ID NO: 208), hu.49 (SEQ ID NO: 209), hu.52 (SEQ ID NO: 210), hu. 13 (SEQ ID NO: 71), hu.64 (SEQ ID NO: 212), rh.56 (SEQ ID NO: 54), hu.2 (SEQ ID NO: 48), hu.18 (SEQ ID NO: 52 ), hu.4 (SEQ ID NO: 47) and hu.67 (SEQ ID NO: 215). These capsid sequences are also found in the Sequence Listing incorporated by reference.

Figures 4A-4D provide an evaluation of the in vitro and in vivo gene transfer efficiency of novel AAV-based primate vectors. The AAV vector is as described in [Gao et al., Proc Natl Acad Sci USA, 99: 11854-11859 (2002, September 3)] having AAV1, 2, 5, 7, 8 and 6 with ch.5, rh. 34, pseudotypes of cy.5, rh.20, rh.8 and AAV9 capsids. As shown in Figure 4A, for in vitro studies, 84-32 cells (293 cells expressing adenovirus serotype E4) seeded in 95-well plates were pseudotyped at a multiplicity of infection (MoI) of 1×10 ⁴ GC per cell. AAVCMVEGFP vector infection. Relative EGFP transduction efficiency was estimated as a percentage of green cells using UV microscopy at 48 hr post-infection and is shown on the y-axis. For in vivo studies, NCR nude mice were administered intraportal (Fig. 4B), intratracheal (Fig. 4C) and intramuscular (Fig. 4D) doses of 1×10 ¹¹ GC per animal (4-6 weeks age) liver ( FIG. 4B ), lung ( FIG. 4C ) and muscle ( FIG. 4D ) to express the vector for the secreted reporter gene A1AT. Serum A1AT levels (ng/ml) were compared at day 28 after gene transfer and are shown on the Y-axis. The X-axis represents the analyzed AAVs and the clades they belong to.

Detailed description of the invention

In any arsenal of vectors for therapy or prophylaxis, choosing a vector source for a desired application requires a variety of different vectors capable of carrying macromolecules to target cells. One problem with the use of AAV as a vector to date has been the lack of a variety of different virus sources. The present invention overcomes this problem by providing clades of AAVs for selection of phylogenetically related (or required for selection of therapeutic regimens), or phylogenetically distinct AAVs and for prediction of function. The present invention also provides novel AAV viruses.

The term "substantial homology" or "substantial similarity" when referring to nucleic acids or fragments thereof means that sequences are aligned when the appropriate nucleotide insertion or deletion is optimally aligned with another nucleic acid (or its complement) at least about 95% to 99% nucleotide sequence identity. Homology is preferred over the full-length sequence, or its open reading frame, or another suitable fragment of at least 15 nucleotides in length. Examples of suitable fragments are described herein.

The terms "sequence identity", "percent sequence identity" or "percent identity" as used herein for nucleic acid sequences mean that the residues in two sequences are the same when aligned for maximum identity. The length of the sequence identity comparison can be the full length of the genome, the full length of the coding sequence of a gene, or fragments requiring at least about 500 to 5000 nucleotides. However, identity in smaller fragments is also desired, for example at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more fragments of nucleotides. Similarly, "percent sequence identity" can be readily determined for amino acid sequences, full length proteins, or fragments thereof. Suitable fragments are at least about 8 amino acids and up to about 700 amino acids in length. Examples of suitable fragments are described in the text.

The term "substantial homology" or "substantial similarity" when referring to amino acids or fragments thereof means that when an appropriate amino acid insertion or deletion is optimally aligned with another amino acid (or its complementary strand), at least About 95% to 99% amino acid sequence identity. Homology is preferred over the full-length sequence, or a protein thereof (eg, cap protein, rep protein), or a fragment thereof of at least 8 amino acids, or more desirably at least 15 amino acids in length. Examples of suitable fragments are described herein.

The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more than 97% identity. Identity can be readily determined by those skilled in the art by means of algorithms and computer programs known to those skilled in the art.

Generally when referring to "identity", "homology" or "similarity" between two different adeno-associated viruses, "identity", "homology" or "similarity" is the reference " The sequence of "comparison" is determined. A "compared" sequence or "alignment" refers to a polynucleic acid sequence or protein (amino acid) sequence that typically contains a correction of missing or additional bases or amino acids compared to a reference sequence. In the Examples, AAV comparisons were performed using published AAV2 or AAV1 sequences as reference points. However, one skilled in the art can easily select another AAV sequence as a reference.

Alignment can be performed using any of a variety of public or commercially available multiple sequence alignment programs. Examples of such programs include "Clustal W", "CAP Sequence Module", "MAP" and "MEME" available through the Internet's World Wide Server. Other sources of such programs are known to those skilled in the art. Alternatively, the VectorNTI utility can be used. Numerous algorithms known in the art, including those included in the programs described above, can also be used to measure nucleotide sequence identity. The use of the program Fasta ^(TM) in GCG Version 6.1 to compare polynucleotide sequences is another example. Fasta ^™ provides alignments and percent sequence identities of the regions of best overlap between query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta ^™ with default parameters (word size 6 and NOPAM coefficients for scoring matrix) provided by GCG version 6.1, which is incorporated herein by reference . Multiple sequence alignment programs can also be applied to amino acid sequences, such as the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically these programs are used with default settings, although those skilled in the art can change these settings as desired. Alternatively, one skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or algorithm as that provided by the referenced algorithm and program. See, eg, JD Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

The term "serotype" distinguishes AAV with a capsid that is serologically distinct from other AAV serotypes. Serological specificity was determined based on the lack of cross-reactivity between antibodies against AAV compared to other AAVs.

Cross-reactivity is usually measured in neutralizing antibody assays. For this assay, adeno-associated virus is used to generate polyclonal sera against specific AAV in rabbits or other suitable animal models. The sera raised against the specific AAV were then tested in this assay for their ability to neutralize the same (homologous) or heterologous AAV. The dilution to achieve 50% neutralization was regarded as the neutralizing antibody titer. Two AAVs are considered to be of the same serotype if the quotient of the reciprocal form of the heterologous titer divided by the homologous titer is less than 16 for the two AAVs. Conversely, two AAVs were considered to be of different serotypes if the ratio of the heterologous titer to the homologous titer in the reciprocal form was 16 or greater.

As defined herein, to form serotype 9, antibodies raised against a selected AAV capsid must not cross-react with any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. In one embodiment, the present invention provides an AAV capsid of a novel serotype identified herein as human AAV serotype 9.

The terms "comprising" and "comprising" used in the specification and claims include other components, elements, integers, steps and the like. Conversely, the term "consisting of" and variations thereof do not include other components, elements, integers, steps and the like.

1. Clade

In one aspect, the invention provides clades of AAV. Clades are based on AAV vp1 amino acid sequence alignments determined as a group of AAVs that are phylogenetically related to each other using a contiguity heuristic with a self-citation value of at least 75% (at least 1000 replicates) and a Poisson-corrected distance measure no greater than 0.05 .

The adjacency algorithm is described in detail in ref. See, eg, M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs can be used to implement the algorithm. For example, MEGA v2.1 The Nei-Gojobori method of program implementation improvement.Using these techniques and computer programs, the sequence of AAV vp1 capsid protein, those skilled in the art can easily determine whether the selected AAV is included in a clade identified herein, Within another clade, or outside these clades.

Although the clades defined here are primarily based on native AAV vp1 capsids, these clades are not limited to native AAV. These clades may include non-native AAV, including (but not limited to) recombinant, modified, chimeric, hybrid, synthetic, artificial, etc., based on AAV vp1 amino acid sequence alignment using a self-citation value of at least 75% (at least 1000 replicates) ) with Poisson-corrected distance measures no greater than 0.05 were identified as phylogenetically related AAVs.

The clades described herein include clade A (represented by AAV1 and AAV6), clade B (represented by AAV2), clade C (represented by AAV2-AAV3 hybrid), clade D (represented by AAV7), clade Clade E (expressed in AAV8) and clade F (expressed in human AAV9). These clades are represented by members of the clades of AAV serotypes described above. AAV1 and AAV6, described above, are members of a single clade (clade A) in which 4 isolates were recovered from 3 human species. The previously described AAV3 and AAV5 serotypes were clearly distinct from each other but were not detected in the screen described herein and were not included in any of these clades.

Clade B (AAV2) and clade C (AAV2-AAV3 hybrid) are the most abundant AAVs found in humans (22 isolates of AAV2 from 12 individuals and 17 isolates of clade C from 8 species individual).

A large number of sequences are grouped in clade D (AAV7) or clade E (AAV8). Interestingly, these clades are ubiquitous across species. Clade D is unique to rhesus and Macaques, with 15 members isolated from 10 different animals. Since clade E is found in humans and nonhuman primates, it is of interest: 9 isolates were recovered from 7 species of humans and 21 isolates were obtained from 9 different nonhuman primates, including Rhesus monkeys, baboons and pigtail monkeys.

The hybrid nature of certain sequences was demonstrated in the other two animals, although in this case all the sequences appeared to have independent And different recombination. None of these recombinants were identified in other animals or subjects.

Because clade C (AAV2-AAV3 hybrid) was identified in 6 different human subjects, recombination even resulted in suitable progeny. On the other hand, in the case of AAV7-AAV8 hybrids, only a few recombination cases were involved in AAV evolution. These recombination scenarios demonstrate that AAV is capable of recombination, resulting in in-frame genes and, in some cases, packaging and/or infectious capsid structures. On the other hand, clade C (the AAV2-AAV3 hybrid clade) is a group of viruses that gain a selective advantage through recombination that allows them to tolerate environmental stress.

A. Clade A (indicated by AAV1 and AAV6):

In another aspect, the present invention provides clade A characterized by containing AAV1 and AAV6 disclosed above. See, eg, International Publication No. WO 00/28061, May 18, 2000; Rutledge et al., JVirol, 72(1):309-319 (January 1998). In addition, this clade contains novel AAVs including, but not limited to, 128.1/hu.43 [SEQ ID NO: 80 and 160], 128.3/hu.44 [SEQ ID NO: 81 and 158], 130.4/hu.48 [SEQ ID NO: 78 and 157] and hu.46 [SEQ ID NO: 82 and 159]. The present invention also provides a modified hu.43 capsid [SEQ ID NO: 236] and a modified hu.46 capsid [SEQ ID NO: 224].

In one embodiment, the capsid of one or more members of the clade shares at least 85%, at least 90%, at least 95%, or at least 97% of the full-length vp1, vp2 or vp3 of the AAV1 and/or AAV6 capsid amino acid identity.

In another embodiment, the invention provides novel AAVs of clade A, provided that none of the novel AAVs contain either AAV1 or AAV6 capsids. These AAVs may include, but are not limited to, AAVs with capsids derived from one or more of the following: 128.1/hu.43 [SEQ ID NO:80 and 160], modified hu.43 [SEQ ID NO:236], 128.3 /hu.44 [SEQ ID NO: 81 and 158], hu.46 [SEQ ID NO: 82 and 159], modified hu.46 [SEQ ID NO: 224] and 130.4/hu.48 [SEQ ID NO: 78 and 157].

B. Clade B (AAV2 clade):

In another embodiment, the invention provides clade B.

This clade is characterized by minimally containing AAV2 as described above and novel AAVs of the present invention, including 52/hu.19 [SEQ ID NO: 62 and 133], 52.1/hu.20 [SEQ ID NO: 63 and 134], 54.5/hu.23 [SEQ ID NO: 60 and 137], 54.2/hu.22 [SEQ ID NO: 67 and 138], 54.7/hu.24 [SEQ ID NO: 66 and 136], 54.1/hu .21 [SEQ ID NO: 65 and 135], 54.4R/hu.27 [SEQ ID NO: 64 and 140], 46.2/hu.28 [SEQ ID NO: 68 and 130], 46.6/hu.29 [SEQ ID NO: 69 and 132], modified hu.29 [SEQ ID NO: 225], 172.1/hu.63 [SEQ ID NO: 171 and 195; GenBank Accession No. AY530624], 172.2/hu.64 [SEQ ID NO GenBank Accession No. AY530625], 24.5/hu.13 [SEQ ID NO: 71 and 129; GenBank Accession No. AY530578], 145.6/hu.56 [SEQ ID NO: 168 and 192], hu.57 [SEQ ID NO: 168 and 192], hu.57 [SEQ ID NO: 71 and 129; ID NO: 169 and 193], 136.1/hu.49 [SEQ ID NO: 165 and 189], 156.1/hu.58 [SEQ ID NO: 179 and 194], 72.2/hu.34 [SEQ ID NO: 72 and 125 GenBank accession number AY530598], 72.3/hu.35 [SEQ ID NO: 73 and 164; GenBank accession number AY530599], 130.1/hu.47 [SEQ ID NO: 77 and 128], 129.1/hu.45 (SEQ ID NO: 76 and 127; GenBank Accession No. AY530608), 140.1/hu.51 [SEQ ID NO: 161 and 190; GenBank Accession No. AY530613] and 140.2/hu.52 [SEQ ID NO: 167 and 191; GenBank Accession No. AY530614 ].

In one embodiment, the capsid of one or more members of the clade has at least 85%, at least 90%, at least 95%, or at least 97% amino acid identity to full-length vp1, vp2, or vp3 of the AAV2 capsid .

In another embodiment, the invention provides novel AAVs of clade B, provided that none of the novel AAVs contain an AAV2 capsid. These AAVs may include, but are not limited to, AAVs with capsids derived from one or more of the following: 52/hu.19 [SEQ ID NOs: 62 and 133], 52.1/hu.20 [SEQ ID NOs: 63 and 134 ], 54.5/hu.23[SEQ ID NO:60 and 137], 54.2/hu.22[SEQ ID NO:67 and 138], 54.7/hu.24[SEQ ID NO:66 and 136], 54.1/hu. 21 [SEQ ID NO: 65 and 135], 54.4R/hu.27 [SEQ ID NO: 64 and 140]; 46.2/hu.28 [SEQ ID NO: 68 and 130]; 46.6/hu.29 [SEQ ID NO:69 and 132]; Modified hu.29[SEQ ID NO:225], 172.1/hu.63[SEQID NO:171 and 195], 172.2/hu.64[SEQID NO:172 and 196]; 24.5/ hu.13 [SEQ ID NO: 71 and 129], 145.6/hu.56 [SEQ ID NO: 168 and 192; GenBank Accession No. AY530618], hu.57 [SEQ ID NO: 169 and 193; GenBank Accession No. AY530619] , 136.1/hu.49 [SEQ ID NO: 165 and 189; GenBank Accession No. AY530612], 156.1/hu.58 [SEQ ID NO: 179 and 194; GenBank Accession No. AY530620], 72.2/hu.34 [SEQ ID NO: 72 and 125], 72.3/hu.35 [SEQ ID NO:73 and 164], 129.1/hu.45 [SEQ ID NO:76 and 127], 130.1/hu.47 [SEQ ID NO:77 and 128, GenBank Accession Nos. AY530610], 140.1/hu.51 [SEQ ID NOs: 161 and 190; GenBank Accession No. AY530613] and 140.2/hu.52 [SEQ ID NOs: 167 and 191; GenBank Accession No. AY530614].

C. Clade C (AAV2-AAV3 hybrid clade)

In another aspect, the present invention provides clade C characterized by AAVs comprising hybrids of AAV2 and AAV3 disclosed above, such as H-6/hu.4, H-2/hu.2 [US Patent Application 2003/0138772 (June 24, 2003)]. In addition, this clade contains novel AAVs including, but not limited to, 3.1/hu.9 [SEQ ID NO: 58 and 155], 16.8/hu.10 [SEQ ID NO: 56 and 156], 16.12/hu.11 [ SEQ ID NO:57 and 153], 145.1/hu.53[SEQ ID NO:176 and 186], 145.6/hu.55[SEQ ID NO:178 and 187], 145.5/hu.54[SEQ ID NO:177 and 188], 7.3/hu.7 [SEQ ID NO: 55 and 150; now deposited with GenBank accession number AY530628], modified hu.7 [SEQ ID NO: 226], 33.4/hu.15 [SEQ ID NO: 50 and 147], 33.8/hu.16 [SEQ ID NO: 51 and 148], hu.18 [SEQ ID NO: 52 and 149], 58.2/hu.25 [SEQ ID NO: 49 and 146], 161.10/hu.60 [SEQ ID NO: 170 and 184], H-5/hu.3 [SEQ ID NO: 44 and 145], H-1/hu.1 [SEQ ID NO: 46 and 144] and 161.6/hu.61 [SEQ ID NO: 174 and 185].

In one embodiment, the capsid of one or more members of the clade shares at least 85%, at least 90%, at least 95% of the full-length vp1, vp2 or vp3 of the hu.4 and/or hu.2 capsids Or at least 97% amino acid identity.

In another embodiment, the present invention provides novel AAVs of clade C (AAV2-AAV3 hybrid clade), provided that none of the novel AAVs contain a hu.2 or hu.4 capsid. These AAVs may include, but are not limited to, AAVs with capsids derived from one or more of the following: 3.1/hu.9 [SEQ ID NOs: 58 and 155], 16.8/hu.10 [SEQ ID NOs: 56 and 156] ], 16.12/hu.11 [SEQ ID NO: 57 and 153], 145.1/hu.53 [SEQ ID NO: 176 and 186], 145.6/hu.55 [SEQ ID NO: 178 and 187], 145.5/hu .54 [SEQ ID NO: 177 and 188], 7.3/hu.7 [SEQ ID NO: 55 and 150], modified hu.7 [SEQ ID NO: 226], 33.4/hu.15 [SEQ ID NO: 50 and 147], 33.8/hu.16 [SEQ ID NO: 51 and 148], 58.2/hu.25 [SEQ ID NO: 49 and 146], 161.10/hu.60 [SEQ ID NO: 170 and 184], H -5/hu.3 [SEQ ID NO: 44 and 145], H-1/hu.1 [SEQ ID NO: 46 and 144] and 161.6/hu.61 [SEQ ID NO: 174 and 185].

D. Clade D (AAV7 clade)

In another embodiment, the invention provides clade D. This clade is characterized as comprising AAV7 as described previously [G. Gao et al., Proc. Natl. Acad. Sci USA, 99: 11854-9 (3 September 2002)]. See SEQ ID NO: 184 for the nucleic acid sequence encoding the AAV7 capsid; see SEQ ID NO: 185 for the amino acid sequence of the AAV7 capsid. In addition, this clade contains many of the previously described AAV sequences, including cy.2, cy.3, cy.4, cy.5, cy.6, rh.13, rh.37, rh.36 and rh.35 [Published US Patent Application No. US2003/0138772A1 (July 24, 2003)]. In addition, the AAV7 clade contains novel AAV sequences including, but not limited to, 2-15/rh.62 [SEQ ID NO:33 and 114], 1-7/rh.48 [SEQ ID NO:32 and 115], 4 -9/rh.54 [SEQ ID NO: 40 and 116] and 4-19/rh.55 [SEQ ID NO: 37 and 117]. The present invention also includes modified cy.5 [SEQ ID NO:227], modified rh.13 [SEQ ID NO:228] and modified rh.37 [SEQ ID NO:229].

In one embodiment, the capsid of one or more members of the clade has at least 85%, at least 90%, at least 95%, or At least 97% amino acid identity.

In another embodiment, the invention provides novel AAVs of clade D, provided that none of the novel AAVs contain cy.2, cy.3, cy.4, cy.5, cy.6, rh.13, rh Any one of .37, rh.36 and rh.35 capsids. These AAVs may include, but are not limited to, AAVs with capsids derived from one or more of the following: 2-15/rh.62 [SEQ ID NO:33 and 114], 1-7/rh.48 [SEQ ID NO: 32 and 115], 4-9/rh.54 [SEQ ID NO: 40 and 116] and 4-19/rh.55 [SEQ ID NO: 37 and 117].

E. Clade E (AAV8 clade)

In one aspect, the invention provides clade E. This clade is characterized by the inclusion of the aforementioned AAV8 [G. Gao et al., Proc. rh.10, rh.25, 29.3/bb.1 and 29.5/bb.2 (Published US Patent Application No. US 2003/0138772A1 (July 24, 2003)).

In addition, novel AAV sequences of this clade include, without limitation, for example, 30.10/pi.1 [SEQ ID NO: 28 and 93], 30.12/pi.2 [SEQ ID NO: 30 and 95], 30.19/pi.3 [SEQ ID NO: 29 and 94], LG-4/rh.38 [SEQ ID NO: 7 and 86], LG-10/rh.40 [SEQ ID NO: 14 and 92], N721-8/rh.43 [SEQ ID NO:43 and 163], 1-8/rh.49[SEQ ID NO:25 and 103], 2-4/rh.50[SEQ ID NO:23 and 108], 2-5/rh.51 [SEQ ID NO:22 and 104], 3-9/rh.52[SEQ ID NO:18 and 96], 3-11/rh.53[SEQ ID NO:17 and 97], 5-3/rh. 57 [SEQ ID NO: 26 and 105], 5-22/rh.58 [SEQ ID NO: 27 and 58], 2-3/rh.61 [SEQ ID NO: 21 and 107], 4-8/rh.64 [SEQ ID NO:15 and 99], 3.1/hu.6[SEQ ID NO:5 and 84], 33.12/hu.17[SEQ ID NO:4 and 83], 106.1/hu.37[SEQ ID NO:10 and 88], LG-9/hu.39 [SEQ ID NO: 24 and 102], 114.3/hu.40 [SEQ ID NO: 11 and 87], 127.2/hu.41 [SEQ ID NO: 6 and 91] , 127.5/hu.42 [SEQ ID NO: 8 and 85], hu.66 [SEQ ID NO: 173 and 197] and hu.67 [SEQ ID NO: 174 and 198]. This clade also includes modified rh.2 [SEQ ID NO:231], modified rh.58 [SEQ ID NO:232], modified rh.64 [SEQ ID NO:233].

In one embodiment, the capsid of one or more members of the clade has at least 85%, at least 90%, at least 95%, or at least 97% amino acid identity to full-length vp1, vp2, or vp3 of the AAV8 capsid . The nucleic acid sequence encoding the AAV8 capsid is shown in SEQ ID NO: 186; the amino acid sequence of the capsid is shown in SEQ ID NO: 187.

In another embodiment, the present invention provides novel AAVs of clade E, provided that none of the novel AAVs contain AAV8.rh.8, 44.2/rh.10, rh.25, 29.3/bb.1 and 29.5/bb .2 Any one of the capsids of [Published US Patent Application No. US2003/0138772A1 (July 24, 2003)]. These AAVs may include, but are not limited to, AAVs with capsids derived from one or more of the following: 30.10/pi.1 [SEQ ID NOs: 28 and 93], 30.12/pi.2 [SEQ ID NOs: 30 and 95 ], 30.19/pi.3 [SEQ ID NO: 29 and 94], LG-4/rh.38 [SEQ ID NO: 7 and 86], LG-10/rh.40 [SEQ ID NO: 14 and 92], N721 -8/rh.43 [SEQ ID NO: 43 and 163], 1-8/rh.49 [SEQ ID NO: 25 and 103], 2-4/rh.50 [SEQ ID NO: 23 and 108], 2-5/rh.51 [SEQ ID NO: 22 and 104], 3-9/rh.52 [SEQ ID NO: 18 and 96], 3-11/rh.53 [SEQ ID NO: 17 and 97], 5-3/rh.57 [SEQ ID NO: 26 and 105], 5-22/rh.58 [SEQ ID NO: 27 and 58], modified rh.58 [SEQ ID NO: 232], 2-3/rh .61 [SEQ ID NO: 21 and 107], 4-8/rh.64 [SEQ ID NO: 15 and 99], modified rh.64 [SEQ ID NO: 233], 3.1/hu.6 [SEQ ID NO:5 and 84], 33.12/hu.17[SEQ ID NO:4 and 83], 106.1/hu.37[SEQ ID NO:10 and 88], LG-9/hu.39[SEQ IDNO:24 and 102], 114.3/hu.40 [SEQ ID NO: 11 and 87], 127.2/hu.41 [SEQ ID NO: 6 and 91], 127.5/hu.42 [SEQ ID NO: 8 and 85], hu.66 [SEQ ID NO: 173 and 197] and hu.67 [SEQ ID NO: 174 and 198].

F. Clade (AAV9 clade)

This clade was identified herein as a new AAV serotype named hu.14/AAV9 [SEQ ID NO: 3 and 123]. In addition, this clade contains other novel sequences, including hu.31 [SEQ ID NO: 1 and 121] and hu.32 [SEQ ID NO: 2 and 122].

In one embodiment, the capsid of one or more members of the clade shares at least 85%, at least 90%, at least 95%, or At least 97% amino acid identity.

In another embodiment, the invention provides novel AAVs of clade F, including but not limited to AAVs having capsids derived from one or more of: hu.14/AAV9 [SEQ ID NOS: 3 and 123 ], hu.31 [SEQ ID NO: 1 and 121] and hu.32 [SEQ ID NO: 1 and 122].

The AAV clades of the invention can be used for a variety of purposes, including providing ready-made libraries of related AAVs for the production of viral vectors and the production of target molecules. Those skilled in the art will appreciate that these clades can also be used as tools for various purposes.

II. New AAV sequences

The present invention provides nucleic acid sequences and amino acids of novel AAV serotypes referred to herein interchangeably as clones hu.14/28.4 and huAAV9. These sequences were used to construct vectors for efficient transduction of liver, muscle and lung. The new AAV and its sequences are also useful for various other purposes. These sequences were submitted by Genbank and given the accession numbers identified herein.

The invention also provides nucleic acid and amino acid sequences of many novel AAVs. As summarized below, many of these sequences included those described above as members of clades.

128.1/hu.43 from clade A [SEQ ID NO:80 and 160, GenBank accession number AY530606], modified hu.43 [SEQ ID NO:236], 128.3/hu.44 [SEQ ID NO:81 and 158, GenBank Accession No. AY530607] and 130.4/hu.48 [SEQ ID NO: 78 and 157, GenBank Accession No. AY530611];

52/hu.19 [SEQ ID NOs: 62 and 133, GenBank Accession No. AY530584], 52.1/hu.20 [SEQ ID NOs: 63 and 134, GenBank Accession No. AY530586], 54.5/hu.23 from clade B [SEQ ID NO: 60 and 137, GenBank accession number AY530589], 54.2/hu.22 [SEQ ID NO: 67 and 138, GenBank accession number AY530588], 54.7/hu.24 [SEQ ID NO: 66 and 136, GenBank accession No. AY530590], 54.1/hu.21 [SEQ ID NO: 65 and 135, GenBank accession number AY530587], 54.4R/hu.27 [SEQ ID NO: 64 and 140, GenBank accession number AY530592], 46.2/hu.28 [SEQ ID NO: 68 and 130, GenBank accession number AY530593], 46.6/hu.29 [SEQ ID NO: 69 and 132, GenBank accession number AY530594], modified hu.29 [SEQ ID NO: 225], 172.1/hu. 63 [SEQ ID NO: 171 and 195] and 140.2/hu.52 [SEQ ID NO: 167 and 191];

3.1/hu.9 [SEQ ID NOs: 58 and 155, GenBank Accession AY530626], 16.8/hu.10 [SEQ ID NOs: 56 and 156, GenBank Accession AY530576], 16.12/hu.11 from clade C [SEQ ID NO: 57 and 153, GenBank accession number AY530577], 145.1/hu.53 [SEQ ID NO: 176 and 186, GenBank accession number AY530615], 145.6/hu.55 [SEQ ID NO: 178 and 187, GenBank accession No. AY530617], 145.5/hu.54 [SEQ ID NO: 177 and 188, GenBank accession number AY530616], 7.3/hu.7 [SEQ ID NO: 55 and 150, GenBank accession number AY530628], modified hu.7[ SEQ ID NO: 226], hu.18 [SEQ ID NO: 52 and 149, GenBank accession number AY530583], 33.4/hu.15 [SEQ ID NO: 50 and 147, GenBank accession number AY530580], 33.8/hu.16 [SEQ ID NO: 51 and 148, GenBank accession number AY530581], 58.2/hu.25 [SEQ ID NO: 49 and 146, GenBank accession number AY530591], 161.10/hu.60 [SEQ ID NO: 170 and 184, GenBank accession number AY530622 ], H-5/hu.3 [SEQ ID NO: 44 and 145, GenBank accession number AY530595], H-1/hu.1 [SEQ ID NO: 46 and 144, GenBank accession number AY530575] and 161.6/hu.61 [SEQ ID NO: 174 and 185, GenBank accession number AY530623];

2-15/rh.62 [SEQ ID NO:33 and 114, GenBank accession number AY530573], 1-7/rh.48 [SEQ ID NO:32 and 115, GenBank accession number AY530561], 4 from clade D -9/rh.54 [SEQ ID NO: 40 and 116, GenBank accession number AY530567], 4-19/rh.55 [SEQ ID NO: 37 and 117, GenBank accession number AY530568], modified cy.5 [SEQ ID NO:227], modified rh.13[SEQ ID NO:228] and modified rh.37[SEQ ID NO:229];

30.10/pi.1 from clade E [SEQ ID NO: 28 and 93, GenBank accession number AY53055], 30.12/pi.2 [SEQ ID NO: 30 and 95, GenBank accession number AY 530554], 30.19/pi. 3 [SEQ ID NO: 29 and 94, GenBank accession number AY530555], LG-4/rh.38 [SEQ ID NO: 7 and 86, GenBank accession number AY 530558], LG-10/rh.40 [SEQ ID NO: 14 and 92, GenBank Accession No. AY530559], N721-8/rh.43 [SEQ ID NO: 43 and 163, GenBank Accession No. AY530560], 1-8/rh.49 [SEQ ID NO: 25 and 103, GenBank Accession No. AY530561], 2-4/rh.50 [SEQ ID NO: 23 and 108, GenBank accession number AY530563], 2-5/rh.51 [SEQ ID NO: 22 and 104, GenBank accession number 530564], 3-9 /rh.52 [SEQ ID NO: 18 and 96, GenBank accession number AY530565], 3-11/rh.53 [SEQ ID NO: 17 and 97, GenBank accession number AY530566], 5-3/rh.57 [SEQ ID NO: 26 and 105, GenBank Accession No. AY530569], 5-22/rh.58 [SEQ ID NO: 27 and 58, GenBank Accession No. 530570], modified rh.58 [SEQ ID NO: 232], 2-3 /rh.61 [SEQ ID NO: 21 and 107, GenBank accession number AY530572], 4-8/rh.64 [SEQ ID NO: 15 and 99, GenBank accession number AY530574], modified rh.64 [SEQ ID NO :233], 3.1/hu.6[SEQ ID NO:5 and 84, GenBank accession number AY530621], 33.12/hu.17[SEQ ID NO:4 and 83, GenBank accession number AY530582], 106.1/hu.37[ SEQ ID NO: 10 and 88, GenBank Accession No. AY530600], LG-9/hu.39 [SEQ ID NO: 24 and 102, GenBank Accession No. AY530601], 114.3/hu.40 [SEQ ID NO: 11 and 87, GenBank Accession number AY530603], 127.2/hu.41 [SEQ ID NO: 6 and 91, GenBank accession number AY530604], 127.5/hu.42 [SEQ ID NO: 8 and 85, GenBank accession number AY530605], hu.66 [SEQ ID NO: 173 and 197, GenBank accession number AY530626], hu.67 [SEQ ID NO: 174 and 198, GenBank Accession No. AY530627] and modified rh.2 [SEQ ID NO: 231];

hu.14/AAV9 [SEQ ID NO: 3 and 123, GenBank accession number AY530579], hu.31 [SEQ ID NO: 1 and 121, AY530596] and hu.32 [SEQ ID NO: 1 and 122, GenBank Accession No. AY530597].

In addition, the present invention provides AAV sequences beyond the scope of the above clades, including rh.59 [SEQ ID NO: 49 and 110], rh.60 [SEQ ID NO: 31 and 120, GenBank accession number AY530571], modified ch. 5 [SEQ ID NO:234] and modified rh.8 [SEQ ID NO:235].

Fragments of the AAV sequences of the invention are also provided. Each of these fragments can be readily used in various vector systems and host cells. The ideal AAV segment has cap proteins, including vp1, vp2, vp3 and hypervariable regions. The method described in US Patent Publication No. US2003/0138772A1 (July 24, 2003) can be used to obtain the rep sequences of the AAV clones identified above, when desired. Such rep sequences include, for example, rep78, rep68, rep52 and rep40 and sequences encoding these proteins. Similarly, other fragments of these clones, including AAV inverted terminal repeats (ITRs), AAV P19 sequences, AAV P40 sequences, rep binding sites, and terminal resolution sites ( terminal resolute site) (TRS). Other suitable fragments will be readily apparent to those skilled in the art.

Capsids and other fragments of the invention can be readily used in a variety of vector systems and host cells. Such fragments may be used alone, in combination with other AAV sequences or fragments, or in combination with elements of other AAV or non-AAV viral sequences. In a particularly desirable embodiment, the vector contains the AAV cap and/rep sequences of the invention.

The AAV sequence and its fragments of the present invention can be used to produce rAAV, and can also be used as an antisense delivery vector, gene therapy vector or vaccine vector. The invention also provides nucleic acid molecules, gene delivery vectors and host cells comprising the AAV sequences of the invention.

Suitable fragments can be determined using the information provided herein.

As described herein, vectors of the invention comprising AAV capsid proteins of the invention are particularly useful in applications where neutralizing antibodies abrogate the efficacy of vectors based on other AAV serotypes, as well as other viral vectors. The rAAV vectors of the present invention are particularly advantageous for repeated administration of rAAV and repeated gene therapy.

These and other embodiments and advantages of the invention are detailed below.

A. AAV serotype 9/hu14 sequence

The present invention provides that the nucleic acid sequences and amino acids of the novel AAV referred to herein as clones hu.14 (formerly known as 28.4) and huAAV9 are interchanged. As defined herein, the new serotype AAV9 refers to an AAV having a capsid that produces antibodies that serologically cross-react with a capsid having the sequence of hu.14 [SEQ ID NO: 123], and the antibody Does not serologically cross-react with antibodies raised against the capsid of any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8.

1. Nucleic acid sequence

The AAV9 nucleic acid sequence of the present invention includes the DNA sequence of SEQ ID NO: 3 consisting of 2211 nucleotides.

The nucleic acid sequence of the present invention also includes a strand complementary to SEQ ID NO: 3, as well as RNA and cDNA sequences corresponding to SEQ ID NO: 3 and their complementary strands. The nucleic acid sequences of the present invention also include natural variants and engineered modifications of SEQ ID NO: 3 and complementary strands thereof. Such modifications include, for example, labeling, methylation, and substitution of one or more natural nucleotides with degenerate nucleotides as are known in the art.

The present invention also includes nucleic acid sequences that are about greater than 90%, preferably at least about 95%, and most preferably at least about 98-99% identical or homologous to SEQ ID NO:3.

The present invention also includes fragments of SEQ ID NO: 3, complementary strands thereof, and cDNAs and RNAs complementary thereto. Suitable fragments are at least 15 nucleotides in length and include functional fragments, ie fragments that have a biological effect. This fragment includes sequences encoding the 3 variable proteins (vp) of the AAV9/HU.14 capsid, which are alternatively spliced variants: vp1 [nt1-2211 of SEQ ID NO: 3], vp2 [SEQ ID NO: 3 about nt411-2211] and vp3 [SEQ ID NO: 3 about nt609-2211]. Other suitable fragments of SEQ ID NO: 3 include fragments containing the initiation codon of the AAV9/HU.14 capsid protein and fragments encoding the hypervariable region of the vp1 capsid protein described herein.

In addition to including the nucleic acid sequences provided in the Figures and Sequence Listing, the invention also includes nucleic acid molecules and sequences designed to express the amino acid sequences, proteins and peptides of the AAV serotypes of the invention. Accordingly, the present invention includes the use of these sequences and/or unique fragments thereof to encode the nucleic acid sequences of novel AAV amino acid sequences and artificial AAV serotypes generated below.

As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-native capsid proteins. Such artificial capsids can be produced by any suitable technique using novel AAV sequences of the invention (e.g., fragments of the vp1 capsid protein) in conjunction with heterologous sequences obtained from another AAV serotype ( known or new), non-contiguous parts of the same AAV serotype, non-AAV viral origin, or non-viral origin. The artificial AAV serotype can be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

2. HU.14/AAV9 amino acid sequence, protein and peptide

The present invention also provides proteins and fragments thereof encoded by the hu.14/AAV9 nucleic acids of the present invention, and hu.14/AAV9 proteins and fragments produced by other methods. As used herein, these proteins include assembled capsids. The invention also includes AAV serotypes produced using the sequences of the novel AAV serotypes of the invention, which are produced using synthetic, recombinant or other techniques known to those skilled in the art. The present invention is not limited to novel AAV amino acid sequences, peptides and proteins expressed by the novel AAV nucleic acid sequences of the present invention, but includes amino acid sequences produced by other methods known in the art, such as by chemical synthesis, other synthetic techniques or other methods, peptides and proteins. The sequence of any of the AAV capsids provided herein can be readily generated using a variety of techniques.

Suitable production techniques are well known to those skilled in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, New York). Alternatively, by the well-known method of solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc., 85: 2149 (1962); Stewart and Young, "Solid Phase Peptide Synthesis" (Freeman, San Francisco, 1969) pp. 27-62) to synthesize peptides. These or other suitable production methods are known to those skilled in the art and do not limit the present invention.

Particularly desirable proteins include the AAV capsid proteins encoded by the nucleotide sequences identified above. The AAV capsid protein includes proteins of 3 alternative splice variants: vpl, vp2 and vp3. Figure 2 provides the full length sequence of vp1. AAV9/HU.14 capsid proteins include vp1 [amino acids (aa) 1-736 of SEQ ID NO: 123], vp2 [about aa138-736 of SEQ ID NO: 123], vp3 [about aa203 of SEQ ID NO: 123 -736] and its functional fragments. Other desirable segments of the capsid protein include constant and variable regions located between hypervariable regions (HVR). Other desirable fragments of the capsid protein include HVR itself.

An algorithm developed to identify regions of sequence divergence in AAV2 yielded 12 hypervariable regions (HVRs), 5 of which overlap or are part of the 4 variable regions described above. [Chiorini et al., J.Virol, 73:1309-19 (1999); Rutledge et al., J.Virol., 72:309-319] identified new AAV serotypes using this algorithm and/or the comparison techniques described herein. HVR. For example, HVRs are located at the following positions: HVR1, aa146-152; HVR2, aa182-186; HVR3, aa262-264; HVR4, aa381-383; HVR5, aa450-474; HVR6, aa490-495; HVR7, aa500-504; HVR9, aa534-555; HVR10, aa581-594; HVR11, aa658-667; and HVRI2, aa705-719 [this numbering system is based on a comparison method using AAV2vp1 as a reference point]. Using the Clustal X program with default settings, or using other commercially available or public comparison programs with default settings, such as described herein, to perform the comparison methods provided herein, those skilled in the art can easily determine the new AAV clothing of the present invention. The corresponding fragment of the shell.

Other desirable fragments of the AAV9/HU.14 capsid protein include amino acids 1-184, amino acids 199-259, amino acids 274-446, amino acids 603-659, amino acids 670-706, amino acids 670-706, SEQ ID NO: 123 of SEQ ID NO: 123 Amino acids 724-736, aa185-198, aa260-273, aa447-477, aa495-602, aa660-669 and aa707-723. In addition, other suitable fragments of the AAV capsid include: aa24-42, aa25-28, aa81-85, aa133-165, aa134-165, aa137-143, aa154-156 according to the numbering of AAV9 [SEQ ID NO: 123] , aa194-208, aa261-274, aa262-274, aa171-173, aa413-417, aa449-478, aa494-525, aa534-571, aa581-601, aa660-671 and aa709-723. Using the ClustalX program with default settings, or using other commercially available or public comparison programs with default settings to perform the comparison methods provided herein, those skilled in the art can easily determine the corresponding fragments of the novel AAV capsids of the present invention.

Other desirable AAV9/HU.14 proteins include rep proteins such as rep68/78 and rep40/52.

Suitable fragments are at least 8 amino acids in length. However, fragments of other desired lengths can readily be used. Such fragments can be produced recombinantly or by other suitable methods, such as chemical synthesis.

The invention also provides other AAV9/HU.14 sequences identified using the sequence information provided herein. For example, with the AAV9/HU.14 sequences provided herein, genome walking technology can be used (Siebert et al., 1995, Nucleic Acid Research, 23: 1087-1088; Friezner-Degen et al., 1986, J. Biol . Chem. 261:6972-6985, BD Biosciences Clontech, Palo Alto, CA) Isolation of infectious AAV9/HU.14. Genome walking techniques are particularly useful for identifying and isolating sequences contiguous to novel sequences identified according to the methods of the present invention. This technique was also used to isolate inverted terminal repeats (ITRs) of the new AAV9/HU.14 serotype based on the novel AAV capsid and rep sequences presented here.

The sequences, proteins and fragments of the invention can be produced by any suitable method, including recombinant production, chemical synthesis or other synthetic methods. Such production methods are known to those skilled in the art and do not limit the invention.

III. Generation of rAAV with novel AAV capsids

The present invention includes novel AAV capsid sequences free of DNA and/or cellular material naturally associated with these viruses. To avoid duplication of all new AAV capsids presented herein, reference is made to the hu.14/AAV9 capsid in this section and in the following sections. However, it should be understood that other novel AAV capsid sequences of the invention can be used in a similar manner.

Another aspect of the invention provides the use of the novel AAV sequences of the invention (including fragments thereof) to generate molecules for the delivery of heterologous genes or other nucleic acid sequences to target cells.

Another aspect of the invention provides the use of the novel AAV sequences of the invention (including fragments thereof) to generate viral vectors for delivery of heterologous genes or other nucleic acid sequences to target cells.

Molecules of the invention comprising AAV sequences comprise any genetic element (vector) that can be delivered to a host cell, such as naked DNA, plasmids, phages, transposons, cosmids, episomes, non-viral Delivery of proteins in vehicles (eg, lipid-based vehicles), viruses, etc. The vector of choice can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated beads, viral infection, and protoplast fusion. Methods for constructing any of the embodiments of the invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering and synthetic techniques. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York.

In one embodiment, the vector of the invention contains, inter alia, a sequence encoding the AAV capsid of the invention or a fragment thereof. In another embodiment, the vector of the present invention contains at least a sequence encoding an AAV rep protein or a fragment thereof. The vectors of the invention may optionally contain AAV cap and rep proteins. In vectors containing both AAV rep and cap, the AAV rep and AAV cap sequences are derived from AAV of the same clade. Alternatively, the rep sequence in the vector provided by the invention is from a different AAV source than the cap sequence. In one embodiment, the rep and cap sequences are expressed from separate sources (eg, separate vectors, or host cells and vectors). In another embodiment, these rep sequences are fused in-frame with cap sequences from different AAV sources to make chimeric AAV vectors off the shelf. The vector of the invention is optionally a vector packaged in an AAV capsid of the invention. These vectors, as well as others described herein, may also contain a minigene comprising the selected transgene flanked by the AAV5'ITR and the AAV3'ITR.

Thus, in one embodiment, the vectors described herein contain a nucleic acid sequence encoding a complete AAV capsid from a single AAV sequence (eg, AAV9/HU.14). Such a capsid may contain amino acids 1-736 of SEQ ID NO:123. Alternatively, these vectors contain sequences encoding artificial capsids comprising one or more fragments of the AAV9/HU.14 capsid fused to heterologous AAV or non-AAV capsid proteins (or fragments thereof). These artificial capsid proteins were selected from discontinuous parts of the AAV9/HU.14 capsid or capsids from other AAVs. For example, rAAV may have a capsid protein comprising one or more AAV9/HU.14 capsid regions selected from vp2 and/or vp3, or fragments thereof selected from: AAV9/HU.14 capsid Shell protein, amino acids 1-184, amino acids 199-259, amino acids 274-466, amino acids 603-659, amino acids 670-706, amino acids 724-738 of SEQ ID NO: 123. In another embodiment, it may be desirable to change the start codon of the vp3 protein to GTG. Alternatively, rAAV may contain one or more of the hypervariable regions of the AAV serotype 9 capsid protein identified herein, or other fragments, including, but not limited to, aa185-198, aa260-273, aa447 of the AAV9/HU.14 capsid -477, aa495-602, aa660-669 and aa707-723. See, SEQ ID NO:123. These modifications may increase expression, yield, and/or improve purification in the expression system of choice, or may be used for another desired purpose (eg, altering tropism or modifying neutralizing antibody epitopes).

The vectors, eg, plasmids, described herein are useful for a variety of purposes, but are particularly useful for generating rAAV comprising a capsid comprising AAV sequences or fragments thereof. These vectors, including rAAV, their elements, constructs and applications are described in detail herein.

In one aspect the present invention provides a method of producing a recombinant adeno-associated virus (AAV) having an AAV serotype 9 capsid or a portion thereof. The method involves culturing a host cell containing a nucleic acid sequence or a fragment thereof encoding an AAV serotype 9 capsid protein as defined herein; a functional rep gene; a small AAV terminal inverted repeat (ITR) and a transgene at a minimum. gene; with sufficient cofactors to package the minigene into the AAV9/HU.14 capsid protein.

The components required for culturing in the host cell to package the AAV minigene into the AAV capsid can be provided to the host cell in trans. Alternatively, the desired components (eg, minigenes, rep sequences, cap sequences, and/or accessory function factors) can be provided by stable host cells engineered to contain one or more of the desired components using methods known to those skilled in the art. Such stable host cells optimally contain the desired components under the control of inducible promoters. However, the desired components can be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. Alternatively, the selected stable host cell may contain a selection component under the control of a constitutive promoter with other selection components under the control of one or more inducible promoters. For example, stable host cells can be generated derived from 293 cells (which contain the El helper function under the control of a constitutive promoter), but contain rep and/or cap proteins under the control of an inducible promoter. Still other stable host cells can be prepared by those skilled in the art.

The minigenes, rep sequences, cap sequences and helper functions required to produce the rAAV of the invention can be delivered to the packaging host cell as any genetic element that conveys the sequences carried thereon. The selected genetic elements can be delivered by any suitable method, including those described herein. Methods for constructing any of the embodiments of the invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering and synthetic techniques. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York. Similarly, methods for producing AAV virions are well known and selection of a suitable method does not limit the invention. See, eg, K. Fisher et al., J. Virol., 70:520-532 (1993) and US Patent No. 5,478,745.

Unless otherwise specified, the AAV ITRs and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9 and the present invention. One of the other new AAV sequences. These ITRs or other AAV components can be readily isolated from AAV sequences using techniques available to those skilled in the art. Such AAV can be isolated or obtained from academic, commercial or public sources (eg, American Type Culture Collection, Manassas, VA). Alternatively, the AAV sequence can be referenced, for example, from a reference or database (e.g., etc.) were obtained by synthesis or other suitable methods.

A. Minigene

Minigenes minimally consist of the transgene and its regulatory sequences with 5' and 3' terminal inverted repeats (ITRs). In a desirable embodiment, the ITR of AAV serotype 2 is used. However, other suitable sources of ITR may also be selected. The minigene is packaged into a capsid protein and delivered to the host cell of choice.

1. Genetically modified

A transgene is a nucleic acid sequence encoding a polypeptide, protein or other product of interest which is heterologous to its flanking vector sequences. The nucleic acid coding sequence is operably linked to regulatory components in a manner that permits transcription, translation and/or expression of the transgene in the host cell.

The composition of the transgene sequence depends on the use of the resulting vector. For example, a certain type of transgenic sequence includes a reporter sequence that, when expressed, produces a detectable signal. Such reporter sequences include, but are not limited to, DNA sequences encoding: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymokinase, green fluorescent protein (GFP), enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins such as CD2, CD4, CD8, influenza hemagglutinin protein and others well known in the art, these substances have high affinity antibodies or can be passed Conventional methods produce such antibodies, as well as fusion proteins comprising an annexin protein suitably fused to the antigen-tagged region of hemagglutinin or Myc.

When these coding sequences are associated with regulatory elements that drive their expression, they can provide a signal that is detected by conventional methods, including enzymatic, radiographic, colorimetric, fluorometric or other spectrometric, fluorescence-activated Cell sorting assays and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, when the marker sequence is the LacZ gene, the presence of a signal-carrying vector is detected by measuring β-galactosidase activity. When the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visualized with a luminometer by the production of color or light.

However, a transgene ideally is a non-marker sequence encoding a product useful in biology or medicine, such as a protein, peptide, RNA, enzyme, dominant negative mutant, or catalytic RNA. Ideal RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNA, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNA. An example of a useful RNA sequence is one that inhibits or eliminates expression of a target nucleic acid sequence in a treated animal. Suitable target sequences generally include tumor targets and viral diseases. See, for examples of such targets, tumor targets and viruses identified in the immunogen-related section below.

Transgenes can be used to correct or ameliorate genetic defects, including defects in which normal genes are expressed below normal levels or in which a functional gene product is not expressed. Alternatively, a transgene may provide a cell with a product not naturally expressed in that cell type or in that host. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide that is expressed in the host cell. The invention also includes the use of multiple transgenes. In some cases, different transgenes can be used to encode individual subunits of a protein, or to encode different peptides or proteins. Ideal when the DNA encoding the protein subunit is bulky, such as for immunoglobulins, platelet-derived growth factor, or dystrophin. To make a cell produce a multi-subunit protein, the cell is infected with a recombinant virus containing each of the different subunits. Alternatively, different subunits of a protein can be encoded by the same transgene. In this case, a single transgene includes DNA encoding each subunit separated by an internal ribosome entry site (IRES). This is desirable when the volume of DNA encoding each subunit is small, eg, the total volume of DNA encoding a subunit and the IRES is less than 5 kilobase pairs. In addition to IRES, DNA can be interrupted by sequences encoding 2A peptides that self-cleavage at the post-translational stage. See, eg, M.L.Donnelly et al., J.Gen.Virol., 78(Part I):13-21 (January 1997); Furler, S et al., Gene Ther, 8(11):864-873 (2001 June); Klump H et al., Gene. Ther., 8(10):811-817 (May 2001). This 2A peptide is significantly smaller than an IRES, making it well suited for use when spacing is the limiting factor. More commonly, when the transgene is large, contains multiple subunits, or when two transgenes are co-delivered, co-administration of rAAV carrying the desired transgene or subunit is permitted so that they concatenate in vivo to form Single vector genome. In such embodiments, a first AAV can carry an expression cassette for expressing a single transgene, while a second AAV can carry an expression cassette for a different transgene that is co-expressed in the host. However, the selected transgene may encode any biologically active or other product, such as that desired for research.

Suitable transgenes can be readily selected by those skilled in the art. The selection of transgenes does not limit the invention.

2. Adjusting element

In addition to the essential elements of the minigene identified above, the vector also contains conventional control elements operatively linked to the transgene in a manner that permits transcription, translation and/or expression of the transgene in a cell using the present Invention produced by plasmid vector transfection or viral infection. As used herein, "operably linked" sequences include expression control sequences contiguous to a gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

Expression control sequences include appropriate transcriptional initiator, terminator, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (ie, the Kozak consensus sequence); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. Many expression control sequences, including native, constitutive, inducible and/or tissue-specific promoters are known and available in the art.

Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see , such as Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1 promoter [ Invitrogen]. Inducible promoters regulate gene expression and may be regulated by exogenously provided compounds, environmental factors such as temperature, or the presence of a specific physiological state, such as the acute phase, a specific differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from various commercial sources including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously provided compounds include zinc-inducible metallothionein (MT) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymeric Enzyme promoter system [International Patent Publication No. WO 98/10088], ecdysone insect promoter [No et al., Proc. Gossen et al., Proc.Natl.Acad.Sci. USA, 89:5547-5551 (1992)], tetracycline inducible system [Gossen et al., Science, 268:1766-1769 (1996), see also Harvey et al., Curr.Opin .Chem.Biol., 2:512-518 (1998)], RU-486 inducible system [Wang et al., Nat.Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432 -441 (1997)] and the rapamycin inducible system [Magari et al., J. Clin. Invest., 100:2856-2872 (1997)]. Other inducible promoters that can be used in the present invention are promoters that can be regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation state of the cell, or only in replicating cells.

In another embodiment the native promoter of the transgene is used. Native promoters are preferred when it is desired that the expression of the transgene mimic native expression. Native promoters can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner or in response to specific transcriptional stimuli. In other embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

Another embodiment of the transgene comprises a gene operably linked to a tissue-specific promoter. For example, if expression in skeletal muscle is desired, a promoter active in muscle should be used. These include promoters from genes encoding skeletal β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, and synthetic muscle promoters that are more active than native promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples of tissue-specific promoters are known from liver (albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); α-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7: 1503-14 (1996)), bone osteocalcin (Stein et al., Mol Biol. Rep., 24: 185- 96 (1997)), bone sialoprotein (Chen et al., J.BoneMiner.Res., 11:654-64 (1996)), lymphocyte (CD2, Hansal et al., J.Immunol., 161:1063-8 (1998 ); immunoglobulin heavy chain; T cell receptor chain), neurons, such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993 )), neurofilament light chain gene (Piccioli et al, Proc. 84(1995)) et al.

A plasmid carrying a therapeutically useful transgene may also optionally contain a selectable marker, or the reporter gene may contain sequences encoding geneticin, hygromycin, or purimycin resistance, among others. Such selectable reporter or marker genes, preferably located outside the viral genome and thus rescued by the methods of the invention, can be used to predict the presence of plasmids, eg ampicillin resistance, in bacterial cells. Other components of the plasmid include an origin of replication. These and other promoters and vector elements can be selected by routine methods and many such sequences are available [see, eg, Sambrook et al., and references cited therein].

For ease of reference herein, the combination of the transgene, promoter/enhancer, and 5' and 3' AAV ITRs is referred to as a "minigene". With the guidance provided herein, such minigenes can be designed by conventional techniques.

3. Delivery of minigenes to packaging host cells

The minigene may be carried on any suitable vector for delivery to the host cell, eg, a plasmid. Plasmids for use in the present invention may be adapted for replication, and optionally integration, in prokaryotic cells, mammalian cells (or both). These plasmids (or other vectors carrying the 5'AAV ITR-heterologous molecule-3'AAV ITR) contain sequences that allow replication of the minigene in eukaryotic and/or prokaryotic cells and selectable markers for these systems. A selectable marker or reporter gene may contain sequences encoding geneticin, hygromycin, or puromycin resistance, among others. These plasmids may also contain certain selectable reporter or marker genes that can be used to indicate the presence of the vector in bacterial cells (eg ampicillin resistance). Other components of the plasmid may contain an origin of replication and an amplicon, for example using the amplicon system of the Epstein Barr virus nuclear antigen. This amplicon system or other similar amplicon components allow high copy episomal replication in cells. Molecules carrying the minigene are preferably transfected into cells where they can exist transiently. Alternatively, the minigene (carrying the 5'AAV ITR-heterologous molecule-3'AAV ITR) can be stably integrated into the host cell genome, either chromosomally or as an episome. In certain embodiments, minigenes may exist in multiple copies, optionally in head-to-head, head-to-tail or tail-to-tail concatemers. Suitable transfection techniques are known and readily used to deliver minigenes to host cells.

When the vector containing the minigene is delivered by transfection, the amount of the vector delivered to about 1×10 ⁴ cells to 1×10 ¹³ cells, or about 1×10 ⁵ cells is generally about 5 μg to 100 μg of DNA, About 10μg-50μg DNA. However, the relative amount of vector DNA delivered to the host cell can be adjusted taking into account factors such as the vector chosen, the method of delivery, and the host cell chosen.

B. Rep and Cap sequences

In addition to the minigene, the host cell contains sequences that drive expression in the host cell of the novel AAV capsid proteins of the invention (or capsid proteins comprising fragments thereof) of the same or cross-complementary origin as the AAV ITRs found in the minigene The rep sequence. AAV cap and rep sequences can be independently obtained from the AAV sources described above and can be introduced into host cells by any means known to those skilled in the art as described above. In addition, when the AAV vector is pseudotyped (e.g., AAV9/HU.14 capsid), different AAV sources (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8) can be provided to encode each major The sequence of the rep protein. For example, the rep78/68 sequence is from AAV2 and the rep52/40 sequence is from AAV8.

In one embodiment, the host cell stably contains the capsid protein under the control of a suitable promoter, such as described above. In this embodiment, the capsid protein is preferably expressed under the control of an inducible promoter. In another embodiment, the capsid protein is provided to the host cell in trans. When the capsid protein is delivered to the host cell in trans, it can be delivered via a plasmid containing the desired sequences to direct expression of the selected capsid protein in the host cell. When the plasmid is delivered to the host cell in trans, the plasmid carrying the capsid protein also preferably carries other sequences required for packaging AAV, such as the rep sequence.

In another embodiment, the host cell stably contains the rep sequence under the control of a suitable promoter, such as those described above. In this embodiment, the primary rep protein is preferably expressed under the control of an inducible promoter. In another embodiment, the rep protein is provided to the host cell in trans. When the rep protein is delivered to the host cell in trans, it can be delivered via a plasmid containing the desired sequence to direct expression of the selected rep protein in the host cell. When the plasmid is delivered to the host cell in trans, the plasmid carrying the capsid protein also preferably carries other sequences required for packaging AAV, such as rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences can be transfected into a host cell on separate nucleic acid molecules and are stably present in the cell as episomes. In another embodiment, the rep and cap sequences are stably integrated into the chromosome of the cell. In another embodiment, the rep and cap sequences are transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection contains from 5' to 3' the promoter, an optional spacer inserted between the promoter and the start of the rep gene sequence, the AAV rep gene sequence, and the AAV cap gene sequence.

The rep and/or cap sequences may optionally be provided on a vector containing the DNA sequence to be introduced into the host cell. For example, a vector may contain an rAAV construct comprising a minigene. The vector may contain one or more genes encoding helper functions such as adenoviral proteins E1, E2a and E4ORF6 and genes for VAI RNA.

The promoter used in this construct is preferably any constitutive, inducible or native promoter known to those skilled in the art or described above. One embodiment uses the AAV P5 promoter sequence. The choice of AAV to provide any of these sequences is not a limitation of the invention.

In another preferred embodiment, the rep promoter is an inducible promoter, such as described above linked to a transgene regulatory element. The preferred promoter for Rep expression is the T7 promoter. A vector containing the rep gene and the cap gene regulated by the T7 promoter is transfected or transformed into cells expressing T7 polymerase either constitutively or inducibly. See International Patent Publication No. WO98/10088, published March 12,1998.

Spacers are optional elements in designing vectors. A spacer is a DNA sequence inserted between the promoter and the ATG start site of the rep gene. The spacer can be of any desired design; ie, it can be a random sequence of nucleotides, or it can encode a gene product, such as a marker gene. Spacers may contain genes that typically include start/stop and polyA sites. A spacer can be a prokaryotic or eukaryotic non-coding DNA sequence, a repetitive non-coding sequence, a coding sequence without transcriptional control or a coding sequence with transcriptional control. Two exemplary sources of spacer sequences are phage ladder sequences or yeast ladder sequences, both commercially available from eg Gibco or Invitrogen, among others. The spacer can be of any size as long as it is sufficient to reduce the expression of rep78 and rep68 gene products and maintain the expression levels of rep52, rep40 and cap gene products at normal levels. Thus, the length of the spacer may be about 10bp-10.0kbp, preferably about 100bp-8.0kbp. To reduce the possibility of recombination, the length of the spacer is preferably less than 2 kbp; however, the present invention is not limited thereto.

While the rep and cap providing molecules may be present transiently in the host cell (i.e., by transfection), one (or both) of the rep and cap proteins and a promoter controlling their expression are preferably stably expressed in the host cell, For example as an episome or by integration into the chromosome of the host cell. Methods used to construct embodiments of the invention are conventional genetic or recombinant engineering techniques, such as those described in the above references. While this specification provides illustrative examples of specific constructs, one of skill in the art can use the information provided herein to select spacers, P5 promoters and other elements (including at least one translational initiation and termination signal) for use with any The addition of polyadenylation sites can be selected so that other suitable constructs can be selected and designed.

In another embodiment of the invention, the rep or cap protein can be stably provided by the host cell.

C. Auxiliary function

In order to package the rAAV of the present invention, host cells also require helper functions. These functionalities may optionally be provided by a herpes virus. The required helper functions are preferably provided by human or non-human primate adenovirus sources, respectively, such as those described above and/or from various sources, including the American Type Culture Collection (ATCC), Manassas, VA (USA ). In a presently preferred embodiment, the host cell is provided with and/or contains the following gene products: an E1a gene product, an E1b gene product, an E2a gene product and/or an E4ORF6 gene product. The host cell may contain other adenoviral genes, such as VAI RNA, but these genes are not required. In preferred embodiments, no other adenoviral genes or gene functions are present in the host cell.

"Adenoviral DNA expressing an Ela gene product"means any adenoviral sequence encoding Ela or any functional portion of Ela. Adenoviral DNA expressing the E2a gene product is similarly defined as adenoviral DNA expressing the E4ORF6 gene product. Also included are any alleles or other modifications of adenoviral genes or functional portions thereof. Such modifications can be introduced intentionally by conventional genetic engineering or mutagenesis techniques, as well as natural allelic variants thereof, to enhance adenovirus function in some manner. Such modifications and methods for manipulating DNA to obtain functionalities of these adenoviral genes are known to those skilled in the art.

The adenoviral E1a, E1b, E2a and/or E4RF6 gene products, as well as any other required helper functions, can be provided using any method for their expression in the cell. Each of the sequences encoding these products can be located on a separate vector, or one or more genes can be located on the same vector. The vector can be any known in the art or disclosed above, including plasmids, cosmids, and viruses. Vectors can be introduced into host cells by any method known in the art or disclosed above, including transfection, infection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated beads, viral infection, and protoplasts body fusion etc. One or more adenoviral genes can be stably integrated into the host cell genome, stably expressed episomally, or transiently expressed. Gene products may all be expressed transiently, expressed episomally, or stably integrated, or some gene products may be expressed stably while others are expressed transiently. In addition, the promoter of each adenoviral gene can be independently selected from a constitutive promoter, an inducible promoter, or a native adenoviral promoter. For example, a promoter may be regulated by a particular physiological state of an organism or cell (ie, by a differentiated state or in replicating or quiescent cells) or by exogenously added factors.

D. Host Cells and Packaging Cell Lines

The host cell itself can be selected from any organism, including prokaryotic (eg, bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Particularly desirable host cells are selected from any mammalian species, including (without limitation) the following cells: A549, WEH1, 3T3, 10T1/2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293 cells (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblasts and hepatocytes from mammals (including humans, monkeys, mice, rats, rabbits and hamsters) and myoblasts. The choice of mammalian species and mammalian cell type (ie, fibroblasts, hepatocytes, tumor cells, etc.) from which the cells are provided is not limiting of the invention. The requirements for the cell used are that it does not carry any adenoviral genes other than E1, E2a, and/or E4ORF6; that it does not contain any other viral genes that could lead to homologous recombination of contaminating virus during rAAV production; and that it infects Or transfect DNA and express the transfected DNA. In a preferred embodiment, the host cell is a cell in which rep and cap are stably transfected.

The host cells used in the present invention are stably transformed with sequences encoding rep and cap and transfected with adenovirus E1, E2a and E4 ORF6 DNA and constructs carrying the above minigenes. Stable rep and/or cap expressing cell lines, such as B-50 (International Patent Application Publication No. WO 99/15685), or the cell lines described in US Pat. No. 5,658,785 may also be used similarly. Another preferred host cell contains minimal adenoviral DNA sufficient to express E4 ORF6. Still other cell lines can be constructed using the novel AAV9cap sequences of the invention.

Preparation of host cells of the invention involves techniques such as assembly of selected DNA sequences. Such assembly can be accomplished using conventional techniques. Such techniques include cDNA and genome cloning well known and described in Sambrook et al. cited above, use of overlapping oligonucleotide sequences from adenovirus and AAV genomes, combination with polymerase chain reaction, synthetic methods and others to provide the desired Suitable methods for nucleotide sequences.

These molecules (plasmids or viruses) can also be introduced into host cells using techniques known to the skilled artisan and discussed in the specification. _In a preferred embodiment, the hybrid adenovirus/AAV vector is introduced into a cell line, such as the human embryonic kidney cell line HEK293 (provides transactivation of E1 protein in a human kidney cell line containing a functional adenovirus E1 gene).

Because no neutralizing antibodies against AAV9/HU.14 have been found in the human population, the AAV9/HU.14-based vectors prepared by those skilled in the art facilitate gene delivery to selected host cells and patients for gene therapy. Other rAAV viral vectors containing the AAV9/HU.14 capsid proteins provided herein can be readily prepared by those skilled in the art using various known techniques. Similarly, one can also prepare other rAAV viral vectors containing AAV9/HU.14 sequences and AAV capsid proteins from other sources.

Those skilled in the art will readily appreciate that the novel AAV sequences of the invention are readily adaptable to these and other viral vector systems for in vitro, ex vivo or in vivo gene delivery. Similarly. Other segments of the AAV genome of the invention can be readily selected by those skilled in the art for use in various rAAV and non-rAAV vector systems. Such vector systems may include, for example, lentivirus, retrovirus, poxvirus, vaccinia virus, and adenovirus systems, among others. The choice of these vector systems does not limit the invention.

Accordingly, the present invention also provides vectors produced using the nucleic acid and amino acid sequences of the novel AAVs of the present invention. Such vectors are used for a variety of purposes, including delivery of therapeutic molecules and use in vaccine dosing regimens. Recombinant AAVs comprising the capsids of the novel AAVs of the invention are particularly desirable for delivery of therapeutic molecules. These or other vector constructs containing the novel AAV sequences of the invention can be used in vaccine dosing regimens, eg, for co-delivery of cytokines or for delivery of the immunogen itself.

IV. Recombinant virus and its application

One of skill in the art can use the techniques described herein to produce rAAV that has a capsid of an AAV of the invention or whose capsid contains one or more fragments of an AAV of the invention. One embodiment uses the full-length capsid of a single AAV, eg, hu.14/AAV9 [SEQ ID NO: 123]. In another embodiment, full-length capsids are produced that contain one or more fragments of a novel AAV capsid of the invention fused to sequences from another selected AAV or to a heterologous (ie, non-contiguous) portion of the same AAV. For example, rAAV may contain one or more novel hypervariable region sequences of AAV9/HU.14. Alternatively, the unique AAV sequences of the invention can be used in constructs containing other viral or non-viral sequences. The recombinant virus may optionally carry an AAV rep sequence encoding one or more AAV rep proteins.

A. Delivery of virus

Another aspect of the present invention provides a method for delivering a transgene to a host, which involves transfecting or infecting selected host cells with a recombinant viral vector produced from the AAV9/HU.14 sequence (or a functional fragment thereof) of the present invention. Methods for delivery are well known to those skilled in the art and do not limit the invention.

In a preferred embodiment, the present invention provides methods for AAV-mediated delivery of a transgene to a host. The method involves transfecting or infecting selected host cells with a recombinant viral vector containing a selected transgene under the control of sequences directing its expression and an AAV9 capsid protein.

Samples from the host can optionally be first assayed for the presence of antibodies against a selected AAV source (eg, one serotype). Various assay formats for detecting neutralizing antibodies are well known to those skilled in the art. The choice of this assay does not limit the invention. See, eg, Fisher et al., Nature Med., 3(3):306-312 (March 1997) and W. C. Manning et al., Human Gene Therapy, 9:477-485 (March 1, 1998). The results of this assay can be used to determine which AAV vectors containing a particular source of capsid protein are preferred for delivery, for example by deleting neutralizing antibodies specific for that source of capsid.

In one aspect of this method, a vector having an AAV capsid protein of the invention may be delivered before or after gene delivery by a vector having a different AAV capsid protein. Thus, gene delivery via rAAV vectors can be used to repeatedly deliver genes to selected host cells. The subsequently administered rAAV vector preferably carries the same transgene as the first rAAV vector, but the subsequently administered vector contains a different source of capsid protein (preferably a different serotype) than the first vector. For example, if the first vector has an AV9/HU.14 capsid protein, subsequently administered vectors may have capsid proteins selected from other AAVs, optionally from another serotype or another clade.

Multiple rAAV vectors can optionally deliver a large transgene or multiple transgenes by co-administering rAAV vectors that concatenate in vivo to form a single vector genome. In such embodiments, a first AAV may carry an expression cassette expressing a single transgene (or a subunit thereof) and a second AAV may carry an expression cassette expressing a second transgene (or a different subunit) for co-expression in the host cell . The first AAV can carry an expression cassette for the first part of the polycistronic construct (e.g., the promoter and transgene, or subunit), and the second AAV can carry the expression cassette for the second part of the polycistronic construct (e.g., the transgene or subunit). subunit and polyA sequence) expression cassette. These two parts of the polycistronic construct concatenate in vivo to form one vector genome that co-expresses the transgene delivered by the first and second AAV. In such embodiments, the rAAV vector carrying the first expression cassette and the rAAV vector carrying the second expression cassette can be delivered in a single pharmaceutical composition. In other embodiments, two or more rAAV vectors are delivered in separate pharmaceutical compositions which may be administered substantially simultaneously or one immediately following the other.

The above-mentioned recombinant vectors can be delivered to host cells according to published methods. rAAV, preferably suspended in a physiologically compatible vehicle, can be administered to a human or non-human mammalian patient. A suitable carrier can be readily selected by those skilled in the art following instructions for delivery of the virus. For example, one suitable carrier includes saline (eg, phosphate buffered saline), which may be formulated in various buffer solutions. Other exemplary vehicles include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrose, agar, pectin, peanut oil, sesame oil and water. The choice of carrier does not limit the invention.

The compositions of the present invention may optionally contain other conventional pharmaceutical components besides rAAV and vehicle, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The vector is administered in an amount sufficient to transfect cells and provide sufficient levels of gene delivery and expression to provide therapeutic benefit without undue adverse effects or with medically acceptable physiological effects, as can be determined by one skilled in the medical art. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via the hepatic artery) or lung), oral, inhalation, intranasal, intragastric, arterial Intraocular, intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral routes of administration. Routes of administration may be combined if desired.

The dosage of the viral vector will depend primarily on factors such as the condition to be treated, the age, weight and health of the patient, and thus may vary from patient to patient. For example, a therapeutically effective human dose of a viral vector is generally about 0.1 mL to 100 mL of a solution containing the viral vector at a concentration of about 1×10 ⁹ to 1×10 ¹⁶ genomes. A preferred human dose for delivery to large organs (eg, liver, muscle, heart, and lungs) may be about 5 x 1010 - ⁵ x ¹⁰¹³ AAV genomes/kg in a volume of about 1-100 mL. A preferred dose for delivery to the eye is about 5 x ¹⁰⁹ - 5 x ¹⁰¹² genome copies in a volume of about 0.1 mL - 1 mL. Dosage may be adjusted to balance the therapeutic benefit against any side effects and may vary depending upon the therapeutic use of the recombinant vector employed. The expression level of the transgene can be monitored to determine the frequency of dosing resulting in a viral vector, preferably an AAV vector containing a minigene. Immunization with the compositions of the invention can optionally be performed using a dosing regimen similar to that described for therapeutic purposes.

Examples of therapeutic and immunogenic products delivered by the AAV-containing vectors of the invention are provided below. These vectors can be used in various therapeutic or vaccination regimens described herein. Furthermore, these carriers may be delivered in combination with one or more other carriers or active ingredients in desired therapeutic and/or vaccination regimens.

B. Therapeutic transgenes

Useful therapeutic products encoded by transgenes include hormones and growth and differentiation factors including, but not limited to, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), Follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony stimulating factor (GCSF), stimulating Erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) ), insulin growth factors I and II (IGF-1 and IGF-II), any of the transforming growth factor alpha superfamily (including TGF alpha), activins, inhibins, or any of the bone morphogenetic proteins (BMPs) One of BMP1-15, heregulin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, aggrin, any of the semaphorin/disintegrin family, netrin-1 and netrin Protein-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog protein, and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the immune system, including (without limitation) cytokines and lymphokines, such as thrombopoietin (TPO), interleukins (IL) IL-1 to IL-25 (such as IL-2, IL- 4. IL-12 and IL-18), monocyte chemical inducer protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and Gamma, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These products include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T Cellular receptors, MHC class I and II molecules, and engineered immunoglobulin and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

Other useful gene products include any of hormone receptors, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. The present invention includes receptors for cholesterol regulation and/or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors and clearance receptor. The invention also includes gene products such as members of the steroid hormone receptor superfamily, including the glucocorticoid receptor and the estrogen receptor. Vitamin D receptor and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum effector factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding protein, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding protein , STAT, GATA-box binding proteins such as GATA-3 and the forkhead protein family of winged helix proteins.

Other useful gene products include carbamoyl synthase 1, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, arginase, fumaryl acetoacetate hydrolase, benzene Alanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porinogen deaminase, cystathionine beta synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA Dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, liver phosphorylase, Phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR) sequence, and dystrophin gene product [eg, small- or micro-dystrophin] . Other useful gene products include enzymes useful in various disorders resulting from enzyme activity deficiency, eg, enzymes useful in enzyme replacement therapy. For example, an enzyme containing mannose-6-phosphate (eg, containing a suitable gene encoding β-glucuronidase (GUSB)) can be used to treat lysozyme storage disease.

Other useful gene products include those for the treatment of hemophilia, including hemophilia B (including factor IX) and hemophilia A (including factor VIII and variants thereof such as heterodimers and B-deleted domains; US Patent Nos. 6,200,560 and 6,221,349). The factor VIII gene encodes 2351 amino acids, and the protein has 6 structural domains, which are designated as A1-A2-B-A3-C1-C2 from amino to carboxy terminus [Wood et al., Nature, 312:330 (1984); Vehar et al. , Nature, 312:337 (1984) and Toole et al., Nature, 342:337 (1984)]. Human Factor VIII is processed intracellularly to produce a heterodimer consisting primarily of a heavy chain containing the Al, A2 and B domains and a light chain containing the A3, Cl and C2 domains. Single-chain polypeptides with heterodimers circulate in plasma as inactive precursors until activated by thrombin cleavage between the A2 and B domains, releasing the B domain and resulting in a heavy chain consisting of the A1 and A2 domains . The B domain is removed in the procoagulant form activated by the protein. Furthermore, in native proteins, the two polypeptide chains ("a" and "b") flanking the B domain bind divalent calcium cations.

In some embodiments, the minigene contains the first 57 base pairs of the Factor VIII heavy chain encoding a 10 amino acid signal sequence and a human growth hormone (hGH) polyadenylation sequence. In an alternative embodiment, the minigene also contains the A1 and A2 domains, and the 5 amino acids N-terminal to the B domain and/or the 85 amino acids C-terminal to the B domain and the A3, Cl and C2 domains. In still other embodiments, the nucleic acids encoding the heavy and light chains of Factor VIII are provided in a single minigene separated by 42 nucleic acids encoding 14 amino acids of the B domain [US Pat. No. 6,200,560].

As used herein, a therapeutically effective amount refers to an amount of AAV vector that produces a sufficient amount of Factor VIII to reduce the time required for blood to clot in a subject. Severe hemophiliacs with factor VIII levels below 1 percent of normal have blood clotting times generally greater than 60 minutes, compared with about 10 minutes for non-hemophiliacs.

The present invention is not limited to any particular Factor VIII sequence. Many natural and recombinant forms of Factor VIII have been isolated and produced. Examples of native and recombinant forms of Factor VIII are found in the patent and scientific literature, including U.S. Patent No. 5,563,045; U.S. Patent No. 5,451,521; U.S. Patent No. 5,422,260; U.S. Patent No. 5,004,803; ; U.S. Patent No. 5,681,746; U.S. Patent No. 5,595,886; U.S. Patent No. 5,045,455; ; U.S. Patent No. 4,886,876; International Patent Publication No. WO 94/11503; WO 87/07144; WO 92/16557; EP 0 270 618; EP 0 182 448; EP 0 162 067; EP 0 786 474; EP 0 533862; EP 0 232112 and EP 0 160 457; Sanberg et al., XXth Int. Congress of the World Fed Of Hemophilia, (1992) and Lind et al., Eur. J. Biochem., 232:19 (1995).

The nucleic acid sequence encoding the aforementioned Factor VIII can be obtained using recombinant methods or by deriving the sequence from a vector known to contain the sequence. In addition, the desired sequence can be isolated directly from cells and tissues containing the sequence using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA [see, eg, Sambrook et al.]. In addition to being cloned, nucleotide sequences can also be produced synthetically. Overlapping oligonucleotides can be prepared by standard methods, from which the complete sequence is assembled and assembled into the complete coding sequence [see, e.g., Edge, Nature, 292:757 (1981); Nambari et al., Science, 223 : 1299 (1984) and Jay et al., J. Biol. Chem., 259: 6311 (1984)].

Furthermore, the present invention is not limited to human Factor VIII. Indeed, the invention should include Factor VIII in animals other than humans, including but not limited to companion animals (e.g., dogs, cats, and horses), livestock (e.g., cattle, goats, and sheep), laboratory Animals, marine animals, large cats, etc.

AAV vectors may contain nucleic acid encoding a fragment of Factor VIII that is itself biologically inactive but improves or restores blood clotting time when administered into a subject. For example, the Factor VIII protein described above contains two polypeptide chains: a heavy chain and a light chain separated by a B domain that is cleaved during processing. As demonstrated in the present invention, co-transduction of recipient cells with the heavy and light chains of Factor VIII results in the expression of biologically active Factor VIII. Since most hemophiliacs have mutations or deletions in only one chain (eg, heavy or light chain), it may be possible to administer only the patient's defective chain to complement the other.

Other useful gene products include non-natural polypeptides, such as chimeric or hybrid polypeptides having native amino acid sequences containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins can be used in certain immunocompromised patients. Other types of non-native genetic sequences include antisense molecules and catalytic nucleic acids, such as ribozymes, that can be used to reduce target overexpression.

Reducing and/or modulating gene expression is particularly desirable for the treatment of hyperproliferative diseases characterized by hyperproliferative cells, such as cancer and psoriasis. Target polypeptides include polypeptides that are produced exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, myc, fyn and translocation genes such as bcr/abl, ras, src, P53, neu, trk and EGRF. In addition to oncogene products as target antigens, target polypeptides for anticancer therapy and protective therapy include the variable regions of antibodies produced by B-cell lymphomas and the variable regions of T-cell receptors in T-cell lymphomas, which T-cell lymphomas are also used in some embodiments as target antigens for autoimmune diseases. Other tumor-associated polypeptides can be used as target polypeptides, such as polypeptides found at higher levels in tumor cells, including polypeptides recognized by monoclonal antibody 17-1A and folate-binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those that treat individuals suffering from autoimmune diseases and disorders by conferring a broad-based protective immune response against autoimmune-related targets, including cellular receptors and the generation of "self-directed Antibody" cells. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis. Each of these diseases is characterized by T cell receptors (TCRs) that bind endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

C. Immunogenic transgene

The AAV vectors of the invention suitably avoid generating an immune response against the AAV sequences contained in the vector. However, these vectors may be formulated in such a way that the transgene carried by the vector is expressed to induce an immune response to the antigen of choice. For example, to promote an immune response, the transgene can be expressed from a constitutive promoter, the vector can be added with helper vectors as described herein and/or the vector can be placed into degenerated tissue.

For example, suitable immunogenic transgenes can be selected from various viral families. Examples of viral families that require an immune response against desired viruses include the picornaviridae of the rhinovirus genus that cause about 50% of the common cold; including polioviruses, coxsackieviruses, echoviruses, and human enteroviruses (such as Hepatitis A virus) of the Enterovirus genus; and Aphthus virus, which mainly causes foot-and-mouth disease in nonhuman primates. Within the picornaviridae, target antigens include VP1, VP2, VP3, VP4 and VPG. Other virus families include the Astrovirus and Caliciviridae. The family Caliciviridae includes the Norwalk group of viruses that are important causes of epidemic gastroenteritis. Another viral family that is expected to target antigens to induce an immune response in humans or non-human animals is the Togaviridae family, which includes the Alphavirus genus, which includes Sindbis virus, Ross River virus, and Venezuelan, Eastern and western equine encephalitis and rubellavirus genera, including rubellaviruses. The Flaviviridae family includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Other target antigens can be generated from Hepatitis C or Coronaviridae, which include many non-human viruses such as Infectious Bronchitis Virus (poultry), Porcine Transmissible Gastroenterovirus (Pig), Porcine Coagulative Encephalomyelitis Virus ), feline infectious peritonitis virus (cats), feline enterocoronavirus (cats), canine coronavirus (dogs), and human respiratory coronaviruses that cause the common cold and/or non-hepatitis A, B, or C and Including the putative cause of Severe Acute Respiratory Syndrome (SARS). Within the Coronaviridae, target antigens include E1 (also known as M or matrix protein), E2 (also known as S or Spike protein), E3 (also known as HE or hemagglutinin-eltiose), glycoprotein (not present in all coronaviruses) or N (nucleocapsid). Other antigens can be targeted against Arteriviridae and Rhabdoviridae, which include the vesivirus genera (eg, herpes stomatitis virus) and lyssaviruses (eg, rabies). Within the Rhabdoviridae, suitable antigens are derived from either the G protein or the N protein. Filoviridae including hemorrhagic fever viruses such as Marburg and Ebola viruses are suitable sources of antigens. Paramyxoviridae include parainfluenza virus type 1, parainfluenza virus type 3, bovine parainfluenza virus type 3, mumps virus (mumps virus), parainfluenza virus type 2, parainfluenza virus type 4, Newcastle disease virus (chicken), rinderpest virus, measles virus, including measles and canine distemper, and pneumoviruses, including respiratory syncytial virus. Influenza viruses are classified within the family Orthomyxoviridae and are a suitable source of antigens (eg, HA protein, N1 protein). The family Bunyaviridae includes the genus Bunyavirus (California encephalitis, La Crosse encephalitis), Phlebovirus (Rift Valley fever), Hantavirus (Pumala is a hemahagin fever virus), Nairobivirus (Nairobi Sheep Disease) and various unnamed bungaviruses. Arenaviridae provide a source of antigens against LCM and Lassa fever viruses. Another source of antigens is the Borneviridae family. The Reoviridae family includes the genera Reovirus, Rotavirus (causes acute gastroenteritis in children), Orbivirus, and Colorado tick fever virus (Colorado tick fever virus, Lebombovirus (human), equine phenovirus encephalopathy virus, bluetongue virus). The family Retroviridae includes the RNA Oncoviridae subfamily, which includes human and veterinary diseases such as Feline Leukemia Virus, HTLVI and HTLVII, the Lentiviridae subfamily (including HIV, Simian Immunodeficiency Virus, Feline Immunodeficiency Virus, Canine Infectious Anemia virus and Foamy virus subfamily). The papovaviridae include the polyomavirinae (BKU and JCU viruses) and the papillomavirinae (associated with the malignant progression of cancer or papilloma). The Adenoviridae family includes viruses (EX, AD7, ARD, O.B.) that cause respiratory disease and/or enteritis. The family Parvoviridae includes feline parvovirus (feline enteritis), feline panleukopenia virus, canine parvovirus, and porcine parvovirus. The family Herpesviridae includes the subfamily Alphaherpesvirinae, which includes the genera Herpes simplex (HSVI, HSVII), Varicellavirus (pseudorabies, varicella-zoster), and the subfamily Betaherpesviridae, which includes cytomegalovirus virus genus (HCMV, murine cytomegalovirus); and the gammaherpesvirus subfamily, which includes the genus Lymphoid Latent Virus, EBV (Burkitt's lymphoma), human herpesviruses 6A, 6B, and 7, Kaposi's sarcoma Related herpesviruses are the simian herpesvirus (B virus), infectious rhinotracheitis virus, Marek's disease virus, and elongate virus. The family Poxviridae includes the subfamily Vertebratepoxvirinae, which includes the genera Orthopoxvirus (variola major (smallpox) and Vaccinia (vaccinia)), Parapoxvirus, Fowlpoxvirus, Capapoxvirus, Lapopoxvirus, Porcinepoxvirus Poxviruses and Entomopoxvirinae. The family Hepadnaviridae includes hepatitis B virus. One unclassified virus which may be a suitable source of antigen is hepatitis delta virus, hepatitis E virus and prions. Another source of antigens is Nipan virus. Other viral sources include avian infectious bursal disease virus and porcine respiratory and reproductive syndrome virus. The Aviridae family includes equine arteritis viruses and various encephalitis viruses.

The invention also includes immunogens for immunizing humans or non-human animals against other pathogens, including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect humans and non-human vertebrates, or from cancer or tumor cells of pathogens. Examples of bacterial pathogens include pathogenic Gram-positive cocci, including pneumococci, staphylococci (with which they produce toxins, such as enterotoxin B), and streptococci. Pathogenic Gram-negative cocci include meningococci and gonorrhoeae. Pathogenic enteric gram-negative bacilli include Enterobacteriaceae; Pseudomonas, Acinetobacter, and Eckenella; Pseudomonas pseudomallei; Salmonella; Shigella; Haemophilus Moraxella sp.; H. ducreyi (causes chancroid); Brucella (brucellosis); Franisella tularensis (causes tularemia); Morinella (plague) and other Yersinia species (Pasteurella species); Streptococcus moniliformis and Helicobacter species; Gram-positive bacilli, including Listeria monocytogenes; Erysipelothrix rhusiopathiae; Diphtheria Corynebacterium diphtheria (diphtheria); cholera; B. anthracis (anthrax); Donovan's disease (granuloma inguinale) and Bartonella. Diseases caused by pathogenic anaerobic bacteria include tetanus, botulism (Clostridium botulimum and its toxins); Clostridium perfringens and its epsilon toxins; other Clostridium species; Tuberculosis, leprosy and other mycobacterial species. Pathogenic treponemal diseases include syphilis; treponemosis: yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by more pathogenic bacteria and pathogenic fungi include gangrene (Burkholderia mallei); actinomycosis; nocardiosis; cryptococcosis, blastomycosis, Histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis and mucormycosis; sporotrichosis; Mycoses; and dermatophytosis. Rickettsial infections include typhus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti), and rickettsial pox Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae, lymphogranuloma venereum, psittacosis, and perinatal chlamydial infections. Pathogenic eukaryotic cells include pathogenic protozoa and helminths and infections resulting from them include: A Miebae, Malaria, Leishmaniasis, Trypanosomiasis, Toxoplasmosis, Pneumocystiscarinii, Trichans, Toxoplasma gondii, Babesiosis, Giardiasis, Trichinella filariasis, schistosomiasis, nematode, trematodes or flukes infection, and tapeworm (tapeworm) infection.

Many of these organisms and/or the toxins they produce are identified by the Centers for Disease Control [(CDC), Department of Health and Human Services, USA] as agents with potential for biological attack. For example, some biological agents, including Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague ), smallpox (smallpox), Franisella tularensis (tularemia), and viral hemorrhagic fevers [filoviruses (e.g., Ebola, Marburg) and arenaviruses [e.g., Lassa virus , Machubo virus]]; Coxiella burnetti (Q fever), Brucella (brucellosis), Burkholderia melioides, now classified as Category B agents Burkholderia mallei (mallei), Burkholderia pseudomallei (malioid), Ricinus communis and its toxin (ricin toxin), perfringens Clostridium perfringens and its toxin (ε toxin), Staphylococcus and its toxin (enterotoxin B), Chlamydia psittaci (psittaci), water safety threats (eg, Vibrio cholerae, Cryptosporidium parvum, typhus fever (Richettsiapowazekii) and viral encephalitis (type A virus, e.g. Venezuelan equine encephalitis, eastern equine Encephalitis, Western Equine Encephalitis); and Nipan virus and Hantavirus, now classified as Category C agents. Additionally, other organisms so classified or classified differently may be identified and/or used for this purpose in the future. Easily It is understood that the viral vectors and other constructs described herein can be used to deliver antigens, viruses, toxins or other by-products of these organisms, which can prevent and/or treat infections or other adverse effects of the use of these biological agents reaction.

Administration of the vectors of the invention to deliver an immunogen against the variable region of T cells elicits an immune response including CTLs to eliminate T cells. In rheumatoid arthritis (RA) several specific variable regions of TCRs involved in the disease have been identified. These TCRs include V-3, V-14, V-17 and V-17. Thus, delivery of a nucleic acid sequence encoding at least one of these polypeptides elicits an immune response targeting T cells involved in RA. In multiple sclerosis (MS) several specific variable regions of TCRs involved in the disease have been identified. These TCRs include V-7 and V-10. Thus, delivery of a nucleic acid sequence encoding at least one of these polypeptides elicits an immune response targeting T cells involved in MS. In scleroderma several specific variable regions of TCRs involved in the disease have been identified. These TCRs include V-6, V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28 and V-12. Thus, delivery of a nucleic acid molecule encoding at least one of these polypeptides elicits an immune response targeting T cells involved in scleroderma.

Thus, the rAAV-derived viral vectors of the invention provide sufficient gene delivery vehicles that are capable of transducing selected genes, in vivo or ex vivo, even in the presence of organisms with neutralizing antibodies against one or more AAV sources. The transgene is delivered to the host cell of choice. In one embodiment, rAAV is mixed with cells ex vivo, the infected cells are cultured using conventional methods, and the transduced cells are reinfused into the patient.

These compositions are especially suitable for gene delivery for therapeutic purposes and for immunization, including the induction of protective immunity. In addition, the compositions of the invention can also be used to produce desired gene products in vitro. For in vitro production, the desired product (e.g., protein) can be obtained from the desired culture after transfecting host cells with rAAV containing the molecule encoding the desired product and culturing the cell culture under conditions that allow expression . Then, the expression product can be purified and isolated if necessary. Suitable transfection, cell culture, purification and isolation techniques are known to those skilled in the art.

The following examples illustrate several aspects and embodiments of the invention.

Example 1 - Computational analysis of primate AAV sequences

A. Collection of Primate Tissue

The source of nonhuman primate tissue has been described [N.Muzyczka, K.I. Berns, in Fields Virology, edited by D.M.Knipe, P.M. Howley (Lippincott Williams & Wilkins, Philadelphia, 2001), vol. Vol, pp. 2327-2359]. Human tissue was collected from surgical procedures or from autopsies or organ donors through two major national human tissue providers, the Cooperative Human Tissue Network (CHTN) and the National Disease Research Interchange (NDRI). organize. The human tissues used in this study consisted of 18 different tissue types, including colon, liver, lung, spleen, kidney, brain, small intestine, bone marrow, heart, lymph nodes, skeletal muscle, ovary, pancreas, stomach, esophagus, Cervix, testes and prostate. Tissue samples were obtained from individuals of different gender, ethnicity (Caucasian, African-American, Asian-Hispanic) and age (23-86 years) groups. Of the 259 analyzed samples from 250 individuals, approximately 28% of the tissues were associated with pathology.

B. Detection and separation of AAV sequences

Total cellular DNA was extracted from human and nonhuman primate tissues as previously described [R.W. Atchison et al., Science 194, 754-756 (1965)]. The molecular predominance and tissue distribution of AAV in humans was determined by signature or full-length cap PCR using primers and conditions similar to those used in the non-human primate assay. The same PCR cloning protocol used for the isolation and characterization of the extended family of non-human primate AAV was employed in AAV isolates from selected human tissues. Briefly, a 3.1 kb fragment containing partial rep and full-length cap sequences was amplified from tissue DNA by PCR and Topo-cloning (Invitrogen). Human AAV clones were initially analyzed by restriction mapping to aid in the identification of AAV sequence diversity, followed by full sequence analysis by SeqWright (SeqWright, Houston, Texas) with 99.9% accuracy. A total of 67 capsid clones (hu.1-hu.67) isolated from human tissues were identified. 86 cap clones from non-human primate tissues were sequenced, of which 70 clones were from rhesus monkeys, 6 clones were from South American macaques, 3 clones were from dolphin-tailed macaques, 2 clones were from baboons, and 5 clones were from chimpanzee.

C. Analysis of AAV sequences

The AAV capsid virus protein (vpl) open reading frame (ORF) of all contiguous sequences was analyzed. Compare the AAV capsid vp1 protein sequence with the ClustalX1.81 ^TM program [HDMayor, JLMelnick, Nature 210, 331-332 (1966)], use BioEdit ^TM [U.Bantel-Schaal, H.Zur Hausen, Virology 134, 52-63 (1984)] software package to generate in-frame DNA alignments. Phylogenetics were inferred using MEGA ^(TM) version 2.1 and TreePuzzle ^(TM) software packages. Using adjacency, maximum parsimony, and maximum likelihood [M. Nei, S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York, 2000); HASchmidt, K. Strimmer, M. Vingron, A. von Haeseler, Bioinformatics 18, 502-4 (March 2002); N. Saitou, M. Nei, Mol Biol Evol 4, 406-25 (July 1987)] algorithm to confirm similar Sequences are clustered.

Clades were then determined from a neighbor-joining phylogenetic tree of all protein sequences. Amino acid distances were estimated using Poisson-correction. Self-citation analysis with 1000 replicates. Sequences were considered monofamily when they had junction nodes within 0.05 genetic distance. A set of sequences from 3 or more sources was considered to be 1 clade. Evidence of recombination for AAV phylogeny was also assessed by sequence analysis. A gap decomposition algorithm [H.J. Bandelt, A.W. Dress, Mol Phylogenet Evol 1, 242-52 (September 1992)] was performed to screen for similarities. The gaps obtained in this way were then further analyzed using the Bootscan algorithm in Simplot software [M.Nei and S.Kumar, "Molecular Evolution and Phylogenetics" (Oxford University Press, New York, 2000)]. restructuring situation. A 100-self-replicated neighbour-joining tree was obtained using a sliding window of 400 nucleotides (10 nucleotides/step). Then, gap resolution and contiguous phylogeny were inferred from the putative recombined fragments. Significant increases in self-priming values, reductions in gaps, and regrouping of hybridizing sequences with their parental sources can be considered criteria for recombination.

Many different cap sequences amplified from 8 different human subjects showed a phylogenetic relationship with AAV2 (5') and AAV3 (3') around a common breakpoint at position 1400 of the Cap DNA sequence, which is consistent with recombination of hybrid viruses and form a consensus. Recombination was detected in the common region of the cap gene from mesenteric lymph node isolates of rhesus monkeys [Gao et al., Proc Natl Acad Sci USA 100, 6081-6086 (13 May 2002)]. The Neib-Gofobori method was performed to select all codons based on the Z-test [R.M. Kotin, Hum Gene Ther 5, 793-801 (July 1994)].

Phylogenetic analysis was performed again to exclude clones positively identified as hybrids. Geese and bird AAVs were included as outgroups in this analysis (I. Bossis, J.A. Chiorini, J Virol 77, 6799-810 (June 2003)). Figure 1 is a neighbor-joining tree; similar relationships were obtained using maximum parsimony and maximum likelihood analyses.

This analysis identified 11 phylogenetic groups (summarized in Table 1). The species origins of the 6 AAV clades and 5 individual AAV clones (or collections of clones) are indicated either numerically or by the origin of sequences recovered in sampling. The total number of sequences collected per class and group is shown in parentheses. The code designation for each of the aforementioned clades is shown in the right column. Rhesus- rhesus monkey; cyno- South American monkey; chimp- chimpanzee; pigtail- dolphin-tailed monkey.

Table 1

Classification of the number of sources (sequences) per species and per clade or clone

As indicated above, since recombination was not performed in the standard phylogenetic algorithm used, sequences whose recombination ancestry was determined were excluded from this analysis in order to build a suitable phylogenetic tree. Neighbor-joining analyzes of all non-recombined sequences are shown side by side with clades that were indeed derived using recombination. Using a different algorithm with the nucleotide sequence as input yielded similar results.

As described in the Examples below, additional experiments were performed to assess phylogenetic affinities with functions detected as serological activity and tropism.

Example 2 - Serological analysis of novel human AAV

As described in the examples above, the last clade obtained was derived from 3 human isolates and did not contain the serotypes mentioned above. Polyclonal antisera against representative members of this clade were generated and serological cross-reactivity between the above serotypes was comprehensively studied. This study demonstrates that the new human clade is serologically distinct from other known serotypes, hence the name clade F (indicated by AAV9).

Rabbit polyclonal antibodies against AAV serotypes 1–9 were generated by intramuscular inoculation of animals with ¹ × 10 genome copies of each AAV vector together with an equal volume of incomplete Freund's adjuvant. Reinjection was given on day 34 to boost antibody titers. Serological cross-reactivity between AAV 1-9 was determined by evaluating the inhibitory effect of rabbit antiserum on transduction of 293 cells with a vector carrying a reporter gene (AAVCMVEGFP carrying enhanced green fluorescent protein) derived from Pseudotypes of capsids from different AAV sources. Transduction of 84-31 cells by AAVCMVEGFP vector was evaluated by UV microscopy. In evaluating the serological relationship between the two AAVs, heterologous and homologous sera were tested for their ability to neutralize each AAV vector. Two AAVs were considered to be different serotypes if the sera were at least 16-fold less capable of neutralizing the heterologous vector in a reciprocal manner than the homologous vector. Neutralization titers were determined as described above (GP Gao et al., Proc Natl Acad Sci US 99, 11854-9 (3 September 2002)).

Table 2

Serological evaluation of new AAV vectors

In addition to the unexpected serological reactivity of the structurally distinct AAV5 and AAV1 serotypes (i.e., heterologous/homologous titer ratios of 1/4 and 1/8 in reciprocal titrations), these data demonstrate distinct Phylogenetic grouping of clones and clades.

The results also indicate that AAVhu.14 has distinct serological properties and has no significant cross-reactivity with antisera raised from any known AAV serotype. Its serological specificity is also confirmed by the unique capsid structure of AAVhu.14, which shares less than 85% of its amino acid sequence with all other AAV serotypes compared in this study. Those findings provided the basis for our designation of AAVhu.14 as a new serotype, AAV9.

Example 3 - Evaluation of primate AAV as a gene delivery vehicle

The biological tropism of AAV was investigated by generating pseudotyped vectors in which recombinant AAV2 genomes expressing GFP or the secreted reporter gene α-1 antitrypsin (A1AT) were used with each strain derived from various clones and used for comparison. Capsids of a representative member of the long-like AAV clade for packaging. For example, data from AAV1 is used to represent clade A, then AAV2 for clade B, Rh.34 for AAV4, AAV7 for clade D, AAV8 for clade E, and AAVHu.14 for clade F. AAV5, AAVCh.5 and AAVRh.8 were compared as individual AAV genotypes.

The transduction efficiency of the vectors was evaluated based on GFP transduction and in vivo in liver, muscle or lung (Fig. 4).

A. In vitro

Vectors expressing enhanced green fluorescent protein (EGFP) were used to test their in vitro transduction efficiency in 84-31 cells and to study their serological characteristics. For functional analysis, in vitro transduction of different AAVCMVEGFP vectors was tested in 84-31 cells seeded in 96-well plates and infected with pseudotyped AAVCMVEGFP vectors at an MOI of ¹ x 104 GC per cell. Using G.Gao et al., Proc Natl Acad Sci U.S. 99, 11854-9 (September 3, 2002) with AAV1, 2, 5, 7, 8 and 6 other new AAVs (Ch.5, Rh .34, Cy5, rh.20, Rh.8, and AAV9) with capsid-pseudotyped AAV vectors. Relative EGFP transduction efficiency was scored as 0, 1, 2 and 3, corresponding to 0-10%, 10-30%, 30-70% and 70-100% of green cells estimated by UV microscopy 48 hours after infection.

B. In vivo

For in vivo studies, human alpha-antitrypsin (A1AT) was selected as a sensitive and quantitative reporter gene in the vector and expressed under the control of the CMV-enhanced chicken beta-actin promoter. Use of the CB promoter enables high levels of tissue-nonspecific and constitutive A1AT gene delivery and enables gene delivery studies in any tissue of interest using the same vector preparation. NCR nude mice aged 4–6 weeks were treated with a novel AAV vector (AAVCBhAlAT) at a dose of ¹ ×1011 genome copies per animal for liver, lung, and muscle by intraportal vein, intratracheal, and intramuscular injections, respectively. Targeted gene delivery. Serum samples were collected at various time points after gene delivery, A1AT concentrations were determined by an ELISA-based assay and scored 0, 1, 2, and 3 relative to different serum A1AT levels 28 days after gene delivery, depending on the route of administration of the vector ( Liver: 0=A1AT<400ng/ml, 1=A1AT 400-1000ng/ml, 2=A1AT 1000-10,000ng/ml, 3=A1AT>10,000ng/ml; Lung: 0=A1AT<200ng/ml, 1= A1AT 200-1000ng/ml, 2=A1AT1000-10,000ng/m1, 3=A1AT>10,000ng/ml; muscle: 0=A1AT<100ng/ml, 1=A1AT100-1000ng/ml, 2=A1AT 1000-10,000ng /ml, 3=A1AT>10,000 ng/ml).

Human AAV, clone 28.4/hu.14 (now named AAV9), transduces liver similarly to AAV8, transduces lung two logs better than AAV5, and transduces muscle better than AAV1, while the other two human Clones 24.5 and 16.12 (hu.12 and hu.13) performed marginally in all 3 target tissues. Clone N721.8 (AAVrh.43) also showed high performance in all 3 tissues.

To further analyze the gene delivery efficiency of AAV9 and rh 43 compared to liver (AAV8), lung (AAV5) and muscle (AAV1 ) candidate markers, dose response experiments were performed. Both novel vectors exhibited at least 10-fold higher gene delivery (efficiency) than AAV1 in muscle, efficiency similar to AAV8 in liver and 2 logs higher than AAV5 in lung.

We demonstrate a panel of AAVs with sufficient gene delivery efficiencies in all 3 tissues that are similar or superior to their candidate markers in each tissue. To date, there are 3 new AAVs in this class, two from rhesus monkeys (rh10 and 43) and one from humans (hou.14 or AAV9). A direct comparison of the relative gene delivery efficiencies of the three AAVs with their candidate markers in murine liver, lung, and muscle suggests that some primate AAVs with the best fitness may have been derived from stringent biological selection as "super" viruses and evolution. These AAVs are well suited for gene delivery.

C. Biological activity characteristics

The unique biological activity signatures of different AAVs were demonstrated to be substantially consistent within members of a set of clones or clades in terms of gene delivery efficiency. However, in vitro transduction cannot predict the efficiency of in vivo gene delivery. An algorithm for comparing biological activity between two different AAV pseudotypes was developed based on relative scores of cumulative assay levels of transgene expression and differences.

The cumulative difference between the in vitro and in vivo gene delivery scores for each pair of AAVs was calculated according to the following formula and is shown in the table (ND = not determined). Cumulative functional difference between vehicles A and B according to score = in vitro (A-B) + lung (A-B) + liver (A-B) + muscle (A-B). The smaller the value, the more similar the functions between AAVs. In gray areas (figures), percent differences of sequences are indicated in bold italics. Percent differences in cap structures were determined by dividing the number of amino acid differences after pairwise deletion of gaps by 750 (the length of the VP1 protein sequence alignment).

AAV1 AAV2 AAV3 Ch.5 AAV4 AAV5 AAV7 AAV8 Rh.8 AAV9 AAV1 0 5 ND 4 4 4 2 4 5 4 AAV2 16.3 0 ND 3 2 4 7 7 6 9 AAV3 13.2 12.3 0 ND ND ND ND ND ND ND Ch.5 15.5 10.5 11.5 0 2 4 6 6 5 8 AAV4 33.7 36.7 34.8 34.9 0 2 7 6 5 8 AAV5 39.1 38.8 38.5 38.4 42.7 0 4 4 3 6 AAV7 14.1 16.7 14.9 15.6 33.2 38.5 0 2 3 2 AAV8 15.6 16.4 14 15.6 33.2 38. 11.6 0 1 2 Rh.8 14.1 15.2 14.3 14.4 33. 39.6 12.1 8.8 0 3 AAV9 17.2 17.3 15.6 14.8 34.5 39.7 17.5 14.3 12.5 0

These studies point to a number of tissues relevant to the study of parvoviruses in humans. The ubiquity of endogenous AAV sequences in a wide range of human tissues suggests that natural infection by this virus group is common. The widespread tissue distribution and frequent detection of viral sequences in the liver, spleen, and gut suggested that transmission was via the gastrointestinal tract and that viremia may be a feature of infection.

The enormous diversity of sequences in humans and nonhuman primates correlates with tropism and serological functionality, suggesting that diversity is driven by real biological stressors, such as immune escape. Clearly, the second most common human clade demonstrating recombination resulting in this diversity is a hybrid of the two aforementioned AAVs.

Topological examination of phylogenetic analysis reveals relationship between viral evolution and its host restriction. The entire genus Dependovirus appears to be derived from avian AAV, consistent with that published by Lukashov and Goudsmit (V.V. Lukashov, J. Goudsmit, JVirol 75, 2729-40 (March 2001)). The AAV4 and AAV5 isolates diverged earlier from the later development of other AAVs. The next important section divides the species into two major monogroups. The first group has clones isolated solely from humans, including clade B, AAV3 clones, clade C, and clade A; the only exception to the species restriction of this group is a single clone of chimpanzee called ch.5. Another single group representing the remaining members of the genus is derived from humans and nonhuman primates. This group includes clade D and rh.8 clones isolated from species other than macaques and the human-specific clade F. The remaining clade within this group (ie, clade E) has members from humans and many nonhuman primates, suggesting that this clade spread beyond species barriers. Interestingly, the capsid structures of clade E members isolated from some humans were essentially identical to those of nonhuman primates, suggesting that little host adaptation had occurred. Biological analysis of AAV8-derived vectors confirmed high levels of gene delivery with broad tissue tropism, consistent with a more promiscuous range of infection and could explain the apparent zoonosis. The extent and efficiency of gene transfer in clade F was even greater, enhancing the possibility of interspecies transmission, which has hitherto been undetected.

The existence of latent AAV widespread transmission throughout humans and nonhuman primates and its apparent propensity for recombination and cross-species barriers raise important questions. Recombination can lead to the emergence of new infectious agents with altered virulence. Evaluation of this possibility is complicated by the fact that the clinical sequelae of AAV infection in primates have yet to be determined. In addition, the high prevalence of AAV sequences in the liver may contribute to the spread of the virus in the population through allogeneic and xenogeneic liver transplantation. Finally, the discovery of endogenous AAV in humans has implications for the use of AAV in human gene therapy. The fact that wild-type AAV is so prevalent in primates without being associated with malignancies suggests that it is not especially oncogenic. In fact, expression of the AAVrep gene was shown to inhibit transformation [P.L. Hermonat, Virology 172, 253-61 (September 1989)].

Example 4 - AAV 2/9 Vectors for Treatment of Cystic Fibrosis Airway Disease

To date, delivery of the CFTR gene to the lung for the treatment of CF airway disease has been limited by poor vector performance and airway epithelial cells posing a significant obstacle to efficient gene delivery. The AAV2 genome packaged in the AAV9 capsid (AAV2/9) was compared to AAV2/5 in various airway model systems.

Nude mice and C57B 1/6 mice were instilled intranasally with a single dose of 50 μl of ¹ ×1011 genome copies (gc) of AAV2/9 expressed at the transcriptional level of the chick β-actin promoter Nuclear targeting under the control of the β-galactosidase (nLacZ) gene or the green fluorescent protein (GFP) gene. After 21 days, lungs and noses were processed for gene expression. Control animals were transduced with AAV2/9-GFP, no LacZ positive cells were seen. AAV2/9-nLacZ was successfully transduced mainly through the airways, and AAV2/5-nLacZ was mainly transduced through the alveoli and almost never through the airways. Both AAV2/5 and AAV2/9 traverse nasal airway epithelium to transduce ciliated and nonciliated epithelial cells.

Epithelial cell-specific promoters are now evaluated for improved targeting to airway cells in vivo. Then, based on the in vivo findings, the gene delivery efficiency of AAV2/9 to human airway epithelial cells was tested. Airway epithelial cells were isolated from human trachea and bronchi and grown at the air-liquid-interface (ALI) on collagen-coated membrane supports. Once the cells were polarized and differentiated, they were transduced with GFP-expressing AAV2/9 or AAV2/5 from the apical and basolateral sides. AAV2/5 and AAV2/9 successfully transduce epithelial cells from the basolateral surface. However, when applied to the tip surface, AAV2/9 transduced 10-fold higher cell numbers than AAV2/5. The gene delivery efficiency of AAV2/9 in the lung and nasal airways of non-human primates was now evaluated.

This experiment demonstrates that AAV2/9 can efficiently transduce the airways of murine lungs and well-differentiated human airway epithelial cells grown in ALI.

Comparison of Embodiment 5-AAV1 (2/1) and AAV9 (2/9) Directly Injected into the Heart of Adult Rats

5×10 ¹¹ AAV2/1 or AAV2/9 particles were injected once into the left ventricle of two adult rats (3 months old).

The results were striking, as significantly higher expression of AAV2/9 vectors than AAV2/1 was observed in the adult rat heart as assessed by the lacZ histochemical method. AAV2/9 also showed better gene delivery efficiency in neonatal mouse hearts.

Example 6 - AAV2/9 Vectors for Gene Therapy of Hemophilia B

In this study, AAV2/9 vectors were shown to be more effective and less immunogenic vectors than traditional AAV sources for liver- and muscle-directed gene therapy for hemophilia B.

For the liver-directed approach, AAV2/9 pseudotyped vectors were evaluated in models of hemophilia in mice and dogs. Following portal vein injection of AAV2/7, 2/8, and 2/9 vectors at 1 × ¹⁰ genome copies (GC)/mouse in immunocompetent hemophilia B mice (in a C57BL/6 background) Long-term supraphysiological levels and shortened activated partial thromboplastin time (aPTT) were achieved in canine factor IX (cFIX, 41-70 μg/ml), where cFIX was posttranscriptionally Expression under the control of a responsive element (WPRE). A 10-fold lower dose (1×10 ¹⁰ GC/mouse) of AAV2/8 vector produced normal levels of cFIX and aPTT time. In dogs with hemophilia B at the University of North Carolina (UNC), administration of the AAV2/8 vector into dogs that had been treated with the AAV2 vector was demonstrated to be successful; a second intraportal injection ( cFIX expression peaked (10 μg/ml) on day 6 after dose = 5×10 ¹² GC/kg), then gradually decreased and stabilized at around 700 ng/ml (16% of normal) throughout the study (1.5 years) . This level was approximately 3-fold higher than in hemophilia B dogs receiving a single injection of AAV2-cFIX at a similar dose. Recently, two naive hemophilia B dogs were injected intraportally with AAV2/8 vector at a dose of 5.25 x ¹⁰¹² GC/kg. After 10 weeks of injection, the cFIX level of one dog (male) reached 30% of the normal level (1.5 μg/ml) and maintained at 1.3-1.5 μg/ml, while the cFIX expression of the second dog (female) was maintained at about About 10% of normal levels. After injection, both the whole blood clotting time (WBCT) and aPTT were shortened, indicating that the antigen is biologically active. After surgery, liver enzymes (aspartate transaminase (SGOT)), alanine transaminase (SGPT) were maintained at normal levels in both dogs. These AAVs were also evaluated for muscle-directed gene therapy in hemophilia B. AAV-CMV-cFIX packaged with 6 different AAV sources was compared in immunocompetent hemophilia B mice (in a C57BL/6 background) after intramuscular injection at a dose of 1×10 ¹¹ GC/mouse - WPRE [AAV carrying cFIX under the control of the CMV promoter and containing WPRE]. Monitor cFIX gene expression and antibody formation. The highest expression was detected in the plasma of mice injected with AAV2/8 vector (1460 ± 392 ng/ml on day 42), followed by injection of AAV2/9 (773 ± 171 ng/ml on day 42) and injection of of AAV2/7 (500±311 ng/ml at day 42). These levels were maintained for 5 months. Surprisingly, the expression level of cFIX with AAV2/1 ranged from 0-253 ng/ml (mean: 66±82 ng/ml). Anti-cFIX inhibitor (IgG) was detected in some AAV2/1 injected mice. cFIX expression levels in these mice correlated well with inhibitor levels. Day 28 samples were further screened for inhibitor formation against all AAVs. Hemophilia B mice showed the highest formation of inhibitors against AAV2/2, followed by AAV2/5 and AAV2/1. Only isolated and low levels of inhibitors were detected in animals injected with AAV2/7, AAV2/8 and AAV2/9. Therefore, the advantage of new AAV serotype 2/9 vectors for muscle-directed gene therapy of hemophilia B is a more efficient and safe vector without eliciting any significant anti-FIX antibody formation.

Example 7 - The new Rh.43 vector of the present invention

A. Comparison of the AAVrh.43-based A1AT expression vector with AAV8 and AAV9 in liver-directed gene delivery in mice

Comparison of hA1AT levels of novel AAVrh.43 vectors belonging to clade E (by phylogenetic analysis) with AAV8 and novel AAV9 following portal vein infusion into mouse livers. More specifically, pseudotyped AAVrh.43, AAV2/8 and AAV2/9 vectors were compared in liver-directed gene delivery in mice. Pseudotyped vectors were intramuscularly administered to 4-6 week old C57BL/6 mice at doses of 1×10 ¹¹ GC, 3×10 ¹⁰ C and 1×10 ¹⁰ GC/animal. Serum samples were collected from animals on day 28 after vector infusion for human α1 antitrypsin (hA1AT) assay.

The data suggest that the new AAVrh.43 vector actually performs similarly to AAV9 in the mouse model.

B. Pseudotyped AAV vector-mediated nuclear-targeted LacZ gene delivery to mouse liver and muscle

The novel AAV9 and AAVrh.43 based vectors of the present invention were compared with AAV1 and AAV2 based vectors. Vector was injected intraportally into the target liver or intramuscularly into the right tibialis anterior muscle of C57BL/6 mice at a dose of 1 x ¹⁰¹¹ GC/mouse. Mice were sacrificed on day 28 after gene delivery and tissues of interest were collected for X-ga1 histochemical staining.

The gene delivery efficiency of the AAVrh.43 vector was confirmed to be close to that of AAV9 but at least 5-fold higher than that of AAV1. AAVrh.43 was further characterized in liver and muscle using the nuclear-targeted LacZ gene as a reporter gene to visualize the extent of gene delivery histochemically.

C. Comparison of AAVrh.43-based A1AT expression vectors with AAV5 in mouse lung-directed gene delivery

The novel rh.43-based vectors of the present invention also exhibit excellent gene delivery capabilities in lung tissue. Pseudotyped vectors were administered intratracheally into the lungs of 4-6 week old C57BL/6 mice at different doses (1×10 ¹⁰ , 3×10 ¹⁰ and 1×10 ¹¹ GC/animal). Serum samples from mice were collected at different time points for hA1AT determination.

The levels of hA1AT after intratracheal instillation of different doses of the vector and AAV5 into the lungs of mice were compared. The data showed that the new vector was at least 100-fold more potent than AAV5 in mouse models.

Example 8 - Novel Human AAV-Based Vectors for Liver and Lung Targeted Gene Delivery

Human clones AAVhu.37, AAVhu.41 and AAVhu.47 were pseudotyped and tested for gene transfer capability in mouse tissues. Pseudotyped AAVCBA1AT vectors with hu.37, hu.41 and hu.47 capsids were prepared using methods described herein and administered to 4-6 week old C57BL/6 mice via intraportal and intratracheal injections. Serum samples from mice were collected on day 14 after vector injection for hA1AT determination according to published techniques. AAVhu.47 belongs to the AAV2 family (clade B) and was isolated from human bone marrow samples. AAVhu.37 and AAVhu.41 were obtained from human testis tissue and human bone marrow samples, respectively. Phylogenetically, they all belong to the AAV8 clade (clade E).

Serum A1AT analysis of injected animals showed that AAV hu.41 and AAV hu.47 underperformed in the 3 tissues tested. However, the gene delivery capacity of the vector derived from AAVhu.37 was similar to that of AAV8 in the liver and AAV9 in the lung.

All publications cited in this specification are incorporated by reference. Although the invention has been described with reference to particularly preferred embodiments, it should be understood that modifications may be made without departing from the inventive concept.

Claims (12)

1. adeno associated virus (i.e. AAV) carrier, it comprises AAV rh.64 capsid, and described AAV rh.64 capsid is by vp1 egg In vain, vp2 albumen and vp3 albumen constitute, described vp1 albumen is by selected from sequence shown in SEQ ID NO:99 or SEQ ID NO:233 Constituting, wherein said AAV also comprises mini-gene, and described mini-gene has AAV end inverted repeat and is operatively connected to regulation The heterologous gene of sequence, described regulation sequence guides described heterologous gene to express in host cell.

2. adeno-associated virus vector as claimed in claim 1, it is characterised in that described capsid is by shown in SEQ ID NO:15 Nucleic acid sequence encoding.

3. the adeno-associated virus vector as according to any one of claim 1-2, it is characterised in that described end inverted repeat comes From the AAV with AAV rh.64 allos.

4. the capsid protein separated, described capsid protein is selected from lower group:

The vp1 capsid protein being made up of the amino acid/11-738 of SEQ ID NO:99, and

The vp1 capsid protein being made up of the amino acid/11-738 of SEQ ID NO:233.

5. a nucleic acid molecules, it comprises the nucleotide sequence of albumen described in coding claim 4.

6. nucleic acid molecules as claimed in claim 5, it is characterised in that the sequence of described nucleic acid is by the nucleoside of SEQ ID NO:15 Acid 1-2217 is constituted.

7. the nucleic acid molecules as described in claim 5 or 6, it is characterised in that described molecule comprises coding AAV capsid protein and merit The AAV sequence of energy property AAV rep albumen.

8. nucleic acid molecules as claimed in claim 7, it is characterised in that described molecule is plasmid.

9. the method producing the recombinant adeno-associated virus (i.e. AAV) containing AAV capsid, it is thin that described method includes cultivating host The step of born of the same parents, described host cell contains: the molecule of (a) coding AAV capsid protein；(b) functional rep gene；C () is contained AAV end inverted repeat (ITRs) and the mini-gene of transgenic；(d) allow to pack into AAV capsid protein described mini-gene Enough miscellaneous function things, wherein said host cell contains the nucleic acid molecules as according to any one of claim 5-8.

10. one kind with institute any one of the adeno-associated virus vector as according to any one of claim 1-3 or claim 5-8 The host cell of the nucleic acid molecules in-vitro transfection stated.

11. 1 kinds of compositionss, it contains the adeno-associated virus vector as according to any one of claim 1-3 or claim 5- Nucleic acid molecules according to any one of 8 and PHYSIOLOGICALLY COMPATIBLE carrier.

12. adeno-associated virus vectors as according to any one of claim 1-3 or compositions as claimed in claim 11, its For molecule is delivered to cell.